Genetic manipulation method

ABSTRACT

A method of inducing transposition in a transgenic embryo, sperm and egg is described, comprising the steps of (a) generating a first adult transgenic organism comprising within its genome one or more copies of a transposon; (b) generating a second adult transgenic organism comprising within its genome one or more copies of a gene encoding a transposase cognate for the transposon and/or a sequence capable of regulating expression of the gene encoding the transposase; (c) crossing the first adult transgenic organism with the second transgenic adult organism to provide a progeny which comprises, in the genome of one or more of its cells, both (i) one or more copies of the transposon and (ii) a gene encoding a transposase cognate for the transposon, wherein the gene encoding the transposase is under the control of one or more inducible regulatory sequences which permit expression of the transposase, and (d) expressing the gene encoding the transposase in the embryo, sperm or egg to cause mobilisation of the transposon within a portion of the tissues or cells of the progeny. Using the method, mobilisation of a transposon can advantageously be induced at predetermined stages of development of an embryo, sperm or egg and the mutated gene of a single cell may be replicated in subsequent cell divisions, resulting in groups of cells which are essentially homogenous for the transposed gene.

RELATED APPLICATIONS

This application is a divisional application which claims priority toUnited States continuation-in-part patent application Ser. No.10/887,636, filed Jul. 9, 2004, which claims priority under 35 U.S.C. §120 to PCT Application Serial PCT/GB03/00065, filed Jan. 9, 2003, whichclaims priority under 35 U.S.C. § 119 to U.S. Provisional Application60/347,107, filed Jan. 9, 2002, Great Britain Application Serial No.0200419.0, filed Jan. 9, 2002, and Great Britain Application Serial No.0211242.3, filed May 16, 2002, the entireties of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for the transfer of geneticinformation in an organism using transposons. In particular, it relatesto a method of inducing genetic modification of cells at a predeterminedstage of development.

BACKGROUND TO THE INVENTION

The development of high through-put DNA sequencing technology, andsophisticated data-capture and computational analysis has resulted inthe sequence determination of entire genomes including Drosophilamelanogaster and Homo sapiens. This has identified novel “predicted”gene sequences but no associated biology ascribing function. Functionalinformation is a prerequisite to delineate which genes may prove to betherapeutic targets for disease management and diagnosis in man.

The identification of individual gene function and the functionalrelationship of genes to disease states is now a pre-occupation of theBiotechnology and Pharmaceutical industry. The identification of diseaserelated genes will allow the development of new drugs or targets fordrug discovery, provide diagnostic or prognostic markers for disease andprovide prescriptive guides for physicians. The latter of these will beparticularly useful in diseases having complex genetics. Where geneticvariation between patients can be measured, personalised medicineprograms can be developed where defined patient responses to drug actionare identified.

Many methods for identifying gene function are being applied, most witha dependency on the comparative analysis of gene structure and geneexpression profiles in healthy and diseased states. The approach isexpensive and time consuming, and the outcomes often subjective, lackinghard evidence relating a variation in gene expression to a functionaldisease related event in vivo. Validation of gene function requiresstudies in animal model systems which directly relate cause (i.e. amutation in a gene sequence, a deletion or an insertion) with ameasurable effect (i.e. behavioural, developmental, metabolic etc.) inthe whole animal.

Gene function studies in mice and other mammals are presently restrictedto:

A) Painstaking mutational analysis of individual genes in “knock-out”mice derived from libraries of embryonic stems cells (ES cells) eachcell containing one or more tagged genes often introduced by viralinfection.

B) The random mutation in vivo of mouse genes by alkylating agents, andsubsequently whole genome sequence analysis to identify multiplemutations.

The knockout approach is valid where a function can be surmised based onsequence homology with closely related genes of known function but thisapproach is time consuming and labour intensive.

The alkylation approach relies entirely on whole genome sequencing toidentify sites of mutation and the cataloguing of changes in previouslydetermined behavioural traits and metabolic read-outs. Identification ofa phenotypic change must then be correlated to one of perhaps onehundred alkylation events in the target mouse genome. The approach isalso time consuming and requires the generation and maintenance of largemouse libraries, and is limited to inbred strains of mice (forcomparative review see Abuin et al. (2002) TIB 20:36-42).

Another method for obtaining mutations is through the introduction ofexogenous DNA into the genome.

Transposons are natural genetic elements capable of jumping ortransposing from one position to another within the genome of a species.Mobilisation of a transposon is dependant on the expression of atransposase enzyme which binds to sequences flanking the transposon DNAleading to the excision of DNA from one position in the genome andreinsertion elsewhere in the genome. Insertion into a gene sequence willlead to a change in gene function which may, in turn, result in ameasurable phenotypic change in the whole organism.

Of the three “classical” model animals, the fly, the worm and the mouse,efficient transposon based insertion methodologies have been developedfor D. melanogaster and for C. elegans. The following class 2 transposonfamilies have been identified: 1) the P family; 2) the hAt family(hobo-Ac-Tam3), (including for example hermes) and the Tcl/marinerfamily (including for example minos, mariner and sleeping beauty).

The introduction of P element mediated transgenesis and insertionalmutagenesis in Drosophila (Spradling & Rubin (1982) Science 218:341-347)transformed Drosophila genetics and formed the paradigm for developingequivalent methodologies in other eukaryotes. However, the P element hasa very restricted host range, and therefore other elements have beenemployed in the past decade as vectors for gene transfer and/ormutagenesis in a variety of complex eukaryotes, including nematodes,plants, mammals, fish e.g. zebrafish and birds.

The use of Drosophila P-elements in D. melanogaster for enhancertrapping and gene tagging has been described; see Wilson et al. (1989)Genes Dev 3:1301; Spradling et al.(1999) Genetics 153:135.

The hobo element of Drosophila melanogaster has been described byGelbart W. M., Blackman R. K., (1989) Prog Nucleic Acid Res Mol Biol36:37-46.

Hermes is derived from the common housefly. Its use in creatingtransgenic insects is described in U.S. Pat. No. 5,614,398, incorporatedherein by reference in its entirety.

Minos, a class 2 transposon and member of the Tcl family of elements,was isolated from D. hydei and has been used for the germ linetransformation of D. melanogaster, C. capitata, and Anopheles stephensi(Loukeris, T. G. et al. (1995) Proc Natl Acad Sci USA 92:9485-9;Loukeris, T. G. et al. (1995) Science 270:2002-5, Catteruccia, F. et al.(2000) Nature 405:959-962) and using transient mobilisation assays ithas also been shown to be active in embryos of D. melanogaster, Aedesaegypti, Anopheles stephensi and Bombyx mori and in cell lines of D.melanogaster, Aedes aegypti, Anopheles gambiae and Spodoptera frugiperda(Catteruccia, F. et al. (2000) Proc Natl Acad Sci USA 97:2157-2162.,Klinakis et al. (2000) EMBO Reports 1:416-421; Shimizu et al. (June2000) Insect Mol Biol 9(3):277-81).

European Patent Application 0955364 (Savakis et al., the disclosure ofwhich is incorporated herein by reference) describes the use of Minos totransform cells, plants and animals. The generation of transgenic micecomprising one or more Minos insertions is also described.

Mariner is a transposon originally isolated from Drosophila mauritiana,but since discovered in several invertebrate and vertebrate species. Theuse of mariner to transform organisms is described in Internationalpatent application WO99/09817.

Salmonid type transposons such as the Sleeping Beauty (SB) transposon, aTcl/mariner-like transposable element reconstructed from fish have beendescribed by Ivics et al. (1997) Cell 91:501-510 and Horie et al. (2001)Proc Natl Acad Sci USA 98, Issue 16, 9191-9196.

International Patent Application WO99/07871 describes the use of the Tcltransposon from C. elegans for the transformation of C. elegans and ahuman cell line.

PiggyBac is a transposon derived from the baculovirus host Trichplusiani. Its use for germ-line transformation of Medfly has been described byHandler et al. (1998) PNAS (USA) 95:7520-5 and U.S. Pat. No. 6,218,185.

In the techniques described in the prior art, the use of the cognatetransposase for inducing transposon jumping (or transposition) isacknowledged to be necessary.

The standard methodology for transposable element mediatedtransformation is by coinjecting into pre-blastoderm embryos a mixtureof two plasmids: one expressing transposase (Helper) but unable totranspose, and one carrying the gene of interest flanked by the invertedterminal repeats of the element (Donor). Transformed progeny of injectedanimals are detected by the expression of dominant marker genes.

PCT/EP01/03341 (WO 01/71019) describes the generation of transgenicanimals using transposable elements. According to this method, thetransposase function is provided by crossing of transgenic organisms,one of which provides a transposon function and the other providing atransposase function in order to produce organisms containing bothtransposon and transposase in the required cells or tissues. The use oftissue specific chromatin opening domains directs transposase activityin a tissue specific manner and gives rise to multiple independenttransposition events in somatic tissues (see Zagoraiou et al. (2001)PNAS 98:11474-11478).

Transpositions can be “tagged” allowing positional changes withincomplex genomes to be rapidly determined and flanking genes determinedby sequence analysis. This allows an immediate link between cause (i.e.an insertional event in a specific gene or regulatory element) andeffect (i.e. a phenotypic or measurable change). However, conventionalmethods of inducing genetic modifications by transposition suffer fromthe disadvantage that the tissue in which transposition has occurredwill be a mosaic of individual cells each with unique transpositions. Asa result, analysis of phenotype results of the transposition event maybe difficult to perform as each transposition event is unique. Thus, amethod of controlling a transposition event so as to provide the samegenetic modification in a number of cells would provide a valuablecontribution to the art.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda method of generating a transgenic progeny and inducing transposition,comprising the steps of:

-   -   (a) generating a first adult transgenic organism comprising        within its genome one or more copies of a transposon;    -   (b) generating a second adult transgenic organism comprising        within its genome one or more copies of a gene encoding a        transposase cognate for the transposon and/or an element capable        of regulating expression of the gene encoding the transposase;    -   (c) crossing the first adult transgenic organism with the second        transgenic adult organism to provide a progeny which comprises,        in the genome of one or more of its cells, both (i) one or more        copies of the transposon and (ii) a gene encoding a transposase        cognate for the transposon, wherein the gene encoding the        transposase is under the control of one or more regulatory        sequences which permit expression of the transposase; and    -   (d) expressing the gene encoding the transposase in the progeny        to cause mobilisation of the transposon within a portion of the        tissues or cells of the progeny.

In one embodiment the gene encoding the transposase is under the controlof an inducible promoter.

Alternatively expressed, the invention thus provides a method ofgenerating a transgenic progeny by transposon mobilisation, comprisingthe steps of:

-   -   (a) providing a progeny which comprises, in the genome of one or        more of its cells, both (i) one or more copies of a transposon        and (ii) one or more genes encoding a transposase cognate for        the transposon, wherein the gene encoding the transposase is        under the control of one or more regulatory sequences which        permit expression of the transposase, and    -   (b) expressing the transposase in the progeny to cause        mobilisation of the transposon within a portion of the tissues        or cells of the progeny.

Suitably, the first adult transgenic organism can be transgenic linescomprising stably integrated “dormant” transposons. Such transgeniclines can be generated using standard genetic modification technologies.Dormant transposons can be induced to transpose through crossing withthe second adult transgenic organism. Accordingly, the inventionprovides a method which can allow the rapid generation of thousands ofmutant progeny, such as mouse mutants.

By “progeny” is meant a double positive transgenic organism thatcomprises in the genome of one or more of its cells both 1) one or morecopies of a transposon and 2) one or more copies of a gene encoding atransposase. A “progeny” as used herein, can be obtained by methods wellknown in the art including, but not limited to 1) co-injection of atransposon and a transposase gene into the fertilized eggs of anorganism; 2) crossing of an organism comprising one or more copies of atransposon with a second organism comprising one or more copies of agene encoding a transposase; 3) injecting a transposase gene into theeggs of an organism comprising one or more copies of a transposon; and4) injecting a transposon into an organism comprising one or more copiesof a gene encoding a transposase.

A “progeny” according to the invention is also made by using ES cells,for example, ES cells that are transfected or infected with one or moreof a transposon and a transposase gene, wherein the ES cells are placedback into an early embryo to obtain the desired modification.Alternatively, a combination of ES technology and egg injectiontechnology is used to obtain a “progeny” of the invention.

In a preferred embodiment, the one or more regulatory sequences whichpermit expression of the transposase are sequences which allow specificexpression of the transposase during germline development. Accordingly,the germ cells of the progeny have transposition events.

FIG. 13 is a schematic diagram showing in vivo transposition in the eggor early embryo. Double positive female progeny are obtained aftercrossing. In one embodiment, a double positive female progeny isproduced by crossing a transposon-positive male and atransposase-positive female, as depicted in FIG. 13. Alternatively, adouble positive male progeny is produced by crossing atransposon-positive female and a transposase-positive male.

Accordingly, in one embodiment, the progeny is a female transgenicorganism resulting from reproduction between the first and secondtransgenic organisms referred to above. In this embodiment, the one ormore regulatory sequences which permit expression of the transposase aresequences which allow specific expression of the transposase duringoogenesis in the female progeny. Thus, transposase expression is inducedupon oogenesis. This, in turn leads to germline transposition eventstaking place in oocytes to generate oocytes having inserted sequences.

In this embodiment, the one or more regulatory sequences which permitexpression of the transposase are derived from regulatory sequences forgenes which are expressed in developing oocytes. Suitable regulatorysequences include those which control the expression of oocytes genessuch as Zp3, Zp1, Zp2, Gdf9, Bmp15, Figla and Mater (see, for example,Rajkovic & Matzuk (2002) Molecular and Cellular Endocrinology 187:5-9).Other suitable regulatory sequences may be derived from the regulatorysequences of Oct-4.

FIG. 14 is a schematic diagram showing in vivo transposition in thesperm. In one embodiment, a double positive male progeny is produced bycrossing a transposon-positive female and a transposase-positive male,as depicted in FIG. 14. Alternatively, a double positive male progeny isproduced by crossing a transposon-positive male and atransposase-positive female.

In one embodiment, in vivo transposition occurs wherein the malecontributes both the transposon and the transposase. Transposition takesplace in the sperm. Mutants are obtained after crossing to a female.

Accordingly, in another embodiment, the progeny is a male transgenicorganism resulting from reproduction between the first and secondtransgenic organisms referred to above. In this embodiment, the one ormore regulatory sequences which permit expression of the transposase aresequences which allow specific expression of the transposase duringspermatogenesis in the male progeny. Thus, transposase expression isinduced upon spermatogenesis. This, in turn leads to germlinetransposition events taking place in spermatocytes to generatespermatocytes having inserted sequences.

In this embodiment, the one or more regulatory sequences which permitexpression of the transposase are derived from regulatory sequences forgenes which are expressed in developing spermatocytes. Suitableregulatory sequences include those which control the expression ofspermatocyte specific mRNAs such as the transcript of the H1t gene(Bartell et al. (August 2000) Biol Of Reproduction 63(2):409-16).

Suitably, the progeny which have transposition events taking place inthe germline are then mated to produce offspring in which thetransposition events can be characterised. A progeny having germlinetransposition can be mated to a normal organism or to an organism which,itself has been generated to have germline transposition. In oneembodiment, the transposase gene comprises a site that allows forelimination of the transposase gene by excision. For example, theinvention provides for a transposon-positive/transposase gene positiveorganism (either male or female), wherein the transposase gene comprisesa lox or FRT site and can be excised by cre or FLP, respectively. In oneembodiment, a male double positive organism is mated to a femaleorganism, wherein the female comprises cre or flp recombinaase in theegg, thereby removing the transposase gene from the early embryo. Inanother embodiment, a male double positive organism is mated to a normalfemale (that is a female that does not comprise cre or flp recombinase).The offspring that results from this mating may not have stabletranspositions. The above described matings are also performed with afemale double positive organism mated with a male.

In a further embodiment the invention, the “progeny” is an embryo.Accordingly, in this embodiment, there is provided a method ofgenerating a transgenic embryo and inducing transposition, comprisingthe steps of:

-   -   a) generating a first adult transgenic organism comprising        within its genome one or more copies of a transposon;    -   b) generating a second adult transgenic organism comprising        within its genome one or more copies of a gene encoding a        transposase cognate for the transposon and/or an element capable        of regulating expression of the gene encoding the transposase;    -   c) crossing the first adult transgenic organism with the second        transgenic adult organism to provide an embryo which comprises,        in the genome of one or more of its cells, both (i) one or more        copies of the transposon and (ii) a gene encoding a transposase        cognate for the transposon, wherein the gene encoding the        transposase is under the control of one or more regulatory        sequences which permit expression of the transposase; and    -   d) expressing the gene encoding the transposase in the embryo to        cause mobilisation of the transposon within a portion of the        tissues or cells of the embryo.

Alternatively expressed, the invention thus provides a method ofgenerating a transgenic embryo by transposon mobilisation, comprisingthe steps of:

-   -   a) providing an embryo which comprises, in the genome of one or        more of its cells, both (i) one or more copies of a transposon        and (ii) one or more genes encoding a transposase cognate for        the transposon, wherein the gene encoding the transposase is        under the control of one or more regulatory sequences which        permit expression of the transposase, and    -   b) expressing the transposase in the embryo to cause        mobilisation of the transposon within a portion of the tissues        or cells of the embryo.

“Embryo” as herein described should be understood to refer to thestructure developing from a single fertilised egg or zygote to the timeof birth or hatching in the case of vertebrates or invertebrates orgermination in the case of plants. Thus, in the context of the presentinvention, “embryo” should be understood to also encompass a mammalianfetus.

FIG. 15 is a schematic diagram showing in vivo transposition usingubiquitous expression. According to this embodiment, either of the maleor female contributes the transposon, and either of the male or femalecontributes the transposase. Transposition takes place in the egg, earlyembryo or sperm.

Mobilisation of a transposon in embryonic cells or tissues may beinduced at any time during the development of the embryo, for example atpredetermined stages of development of an embryo. By inducingtransposition during such stages of development, the mutated gene of asingle cell may be replicated in subsequent cell divisions, resulting ina group or groups of cells which are essentially homogeneous for thetransposed gene, only if the transposase is no longer active (forexample, if the transposase gene has been excised as described herein).Thus the transposed gene may be present in some or all of the cells of aparticular tissue or group of tissues. The invention thus enables thegeneration of transgenic embryos and organisms comprising one or moreclonal populations of cells homogeneous for one or more individualmutations.

Thus, a second aspect of the invention provides a method of generating atransgenic organism having a plurality of cells or tissues homogeneousfor a gene modified by transposon mobilisation, the method comprisinggenerating a transgenic embryo and inducing transposition thereinaccording to the method of the first aspect of the invention.

By enabling the regulation of transposition at different times duringdevelopment, the method of the present invention also increases thelikelihood of genome-wide transposition since chromatin domainsaccessible to transcriptional complexes and, in all probability,transposition events vary in different cell tissue types at differenttimes during embryonic development and moreover in adult life and inabnormal growth situations such as tumours.

The likelihood of achieving transposition in particular regions of thegenome may be increased further by the use of chromatin opening domains,for example ubiquitously-acting chromatin opening elements (UCOEs)(PCT/GB99/02357 (WO 0005393)), locus control regions (LCRs) (Fraser, P.& Grosveld, F. (1998) Curr Opin Cell Biol 10:361-365), CpG islands orinsulators to control expression of the transposon and/or the geneencoding the transposase.

In preferred embodiments of the invention, the transposon and/or thegene encoding the transposase and/or a sequence regulating expression ofthe gene encoding the transposase are incorporated within chromatinopening domains to increase the likelihood of achieving transposition ina target tissue of the embryo, thus enabling the generation of apopulation of cells of that tissue homogeneous for a transpositionevent. For example, where it is desired to induce transposition in adefined tissue, the transposon and/or gene encoding the transposaseand/or a sequence regulating expression of the gene encoding thetransposase are incorporated within a locus control region, conferringtissue specific control on the expression of a transgene in that tissue.Where it is desired to induce transposition in a tissue for whichspecific LCRs are not available and/or where it is desired to induceearly induction of transposition in a particular tissue, the transposonand/or the gene encoding the transposase and/or a sequence regulatingexpression of the gene encoding the transposase may be incorporatedwithin a UCOE. In particularly preferred embodiments of the invention,both the transposon and the gene encoding the transposase areincorporated within chromatin opening domains. This advantageouslyenhances the efficiency of transposition in particular loci throughoutthe genome during embryo development, enabling the production of one ormore populations of cells homogeneous for a particular transpositionevent.

Thus the invention further provides in a third aspect a method ofgenerating a transgenic embryo and inducing transposition, comprisingthe steps of:

-   -   (a) generating a first adult transgenic organism comprising        within its genome one or more copies of a transposon;    -   (b) generating a second adult transgenic organism comprising        within its genome one or more copies of a gene encoding a        transposase cognate for the transposon and/or an element capable        of regulating expression of the gene encoding the transposase;    -   (c) crossing the first adult transgenic organism with the second        transgenic adult organism to provide an embryo which comprises,        in the genome of one or more of its cells, both (i) one or more        copies of the transposon and (ii) a gene encoding a transposase        cognate for the transposon, wherein the gene encoding the        transposase is under the control of one or more regulatory        sequences which permit expression of the transposase and wherein        the transposon and/or the gene encoding the transposase and/or a        gene regulating expression of the gene encoding the transposase        lie within a chromatin opening domain; and    -   (d) expressing the gene encoding the transposase in the embryo        to cause mobilisation of the transposon within a portion of the        tissues or cells of the embryo.

In preferred embodiments of the invention, the embryo is produced bycrossing a first organism, which is a transgenic organism comprising oneor more copies of the transposon, with a second organism, which is atransgenic organism which comprises, in its genome one or more copies ofthe regulatable gene encoding a cognate transposase. In an alternativeembodiment, the embryo may be produced by crossing a first organism,which is a transgenic organism comprising one or more copies of both thetransposon and the gene encoding the cognate transposase with a secondorganism comprising one or more copies of regulatory elements necessaryto permit transposase expression.

In a preferred embodiment, the transposon and the gene encoding thetransposase may be provided as a single construct such that the geneencoding the transposase is disrupted when the transposon mobilises,thus limiting further mobilisation of the transposon. This may beachieved by placing one of the inverted repeats of the transposon in anintron which interrupts the transposase gene such that the transposasegene is disrupted when the transposon is mobilised.

This vector enables a single cross step to be used to generate atransgenic organism that contains regulator, transposase gene andtransposon. Further, transposition leads to complete inactivation of thetransposase source, resulting in stability of the new insertion even inthe presence of inducer. FIG. 1 illustrates schematically the use ofsuch a vector in the present invention.

The incorporation of Cre/lox functions (details of which are reviewed inSauer (1993) Methods of Enzymology 225:90-900) or Flp/frt functions(Farley et al. (2000) Genesis 28:106-110; Schaft et al. (2001) Genesis31:6-10) and different transposon/transposase combinations may also beused to eliminate primary transposase function. In further embodimentsof the invention, however, the transposase gene is not destroyed ontransposition, thus allowing further transposon mobilisation on, forexample, administration of inducer.

In methods of the invention, transposition may be induced using anysystem known to the skilled person. Transposition may be induced byinduction of transposase gene expression via application of anendogenous substance or via operation of an endogenous signal, such as adevelopmental regulated signal.

The one or more regulatory sequences of which the gene encoding thetransposase is under the control may be inducible regulatory sequences.For example, suitable induction systems include tet based systems, thelac operator-repressor system, ecdysone based systems and oestrogenbased systems, details of which are provided infra. Exogenous inducersmay be provided in any convenient fashion, e.g. by injection to thematernal animal or embryo or as an additive to the food or water supplyto the maternal animal. Transposition may be induced at one or moretimes during embryo development. Thus inducers may be administered onlyonce or repeatedly during one or more stages of development.

In alternative embodiments of the invention, expression of the geneencoding the transposase may be induced in response to a gene regulatorysignal produced at a particular stage of embryo development. Suchcontrol may be achieved by placing the gene encoding the transposaseunder the control of a gene regulatory sequence such as a developmentalregulated sequence or promoter, for example, a developmental regulatedspecific promoter responsive to a particular gene regulatory signal suchas a transiently expressed development regulated protein. Where the geneencoding the transposase is under such control, expression of the geneencoding the transposase will only occur when the gene regulatorysignal, for example a transiently expressed developmental regulatedprotein, is produced, or, alternatively, when such a signal protein isintroduced to the embryo, for example by injection or in the feed of thematernal animal.

Using the methods of the invention, the timing of the expression of thegene encoding the transposase and hence the timing of the induction oftransposition in an embryo may be controlled.

In embodiments where it is desired to tightly control the duration andeffect of expression of the transposase gene and thus further restrictthe timing of transposition, the gene encoding the transposase may beprovided within the same construct as the transposon such that, ontransposon mobilisation, the gene encoding the transposase is disrupted,preventing the production of further transposase and thus limitingfurther transposition.

By thus selecting the time of induction, mobilisation of transposons maybe induced at a predetermined stage of embryo development. For example,transposition may be induced at very early stages of development such asat the zygote stage, a four cell embryo, sixty-four cell embryo etc. orat later stages of development.

In one embodiment, induction of transposition by placing the transposaseunder the control of one or more regulatory sequences that will drivetransposase gene expression in the early fertilised egg e.g. at the twoor four cell stage is desirable. Suitable regulatory sequences forcontrolling transposase expression at this stage may be derived from theregulatory sequences of genes whose expression is activated at thisstage. Such genes include Oct-4 (Kirchof et al. (December 2000) BiolReprod 63(6):1698-705), and maternal effect genes such as Zp1, Zp2,Gdf9, Bmp15, Figla and Mater. Other suitable genes include hsp70.1(Bevilacqua et al. (April 2000) Development 127(7):1541-51).

In another embodiment, induction of transposition by placing thetransposase under the control of one or more regulatory sequences thatwill drive transposase gene expression in the early sperm is desirable.Suitable regulatory sequences for controlling transposase expression atthis stage may be derived from the regulatory sequences of genes whoseexpression is activated at this stage. Such genes include the earlyhistone sperm specific promoter (for example the H1t promoter (see Wolfeet al., 2003, Biology of Reproduction, 68:2267-2273)). In one embodimentof the invention a transposase is integrated into the endogenous H1tlocus. The invention therefore encompasses all of the regulatorysequences present in the endogenous H1t locus. The invention alsocontemplates any regulatory sequence that will drive transposaseexpression in the sperm that is known in the art.

Depending on the stage of development, cells in which transposition hasoccurred may divide with further rounds of cell divisions resulting in apopulation of cells homogeneous for the initial transposition event.Where transposition has been induced more than once, a population ofcells may be homogeneous for each of two or more transposition events.Thus, depending on the stage of development, an insertional event may bepresent in populations of cells within one or more tissues, completetissues or groups of tissues. The precise nature of the insertionalevent will determine whether it will influence functional geneexpression in some or all embryonic and adult tissues. Thus, geneexpression patterns of modified genes can be monitored during embryodevelopment and in adult cells and tissues.

Moreover, similar populations of cells homogeneous for an initialtransposition event can also be generated in rapidly growing adult cellsand tissue derived from stem cells, typically during tissue regenerationor during cell and tissue maintenance. Examples of such cells andtissues include but are not limited to cells of the gut lining, theliver and blood, which are subject to rapid turnover and/or regenerationfrom stem cells in the adult. Similarly, populations of cellshomogeneous for a transposition event may be generated in tumours. Themethods of the invention may be adapted to provide populations of cellshomogeneous for an initial transposition event in such adult cells.

Thus, in further embodiments of the invention, step (d) of the first orthird aspect of the invention may be adapted by expressing the geneencoding the transposase in a neonatal, young adult or adult organism tocause mobilisation of the transposon within a portion of the tissues orcells of the organism instead of or, preferably, in addition toexpressing the transposase in the embryo. Thus, the method of theinvention enables the generation of a population of cells homogeneousfor a transposition event induced at a predetermined stage ofdevelopment in a neonate, young adult or adult organism. In suchembodiments, the transposon or the gene encoding the transposase ispreferably under the control of a locus control region to enable tissuespecific control of the transgene. For example, where it is desired toinduce transposition in liver cells, the gene encoding the transposasemay be under the control of a locus control region associated withexpression in liver cells.

Where transposition events are induced in the early development of thezygote, it is possible to develop ES cell lines having transpositions.These ES cell lines can be sequenced, characterised and stored forfuture use.

The generation of genetic mutations in transgenic organisms as a resultof transposon insertion according to the invention may give rise tonovel phenotypic variations in the organisms. Using the methods of theinvention, transgenic embryos may be produced in which one or moreclusters of cells or tissues are each homogeneous for a differenttransposition event, each of which may or may not have a phenotypiceffect. Thus using the methods of the invention, embryos and adultsdeveloped therefrom comprising one or more clusters or groups of cellseach displaying a phenotypic variation compared to the phenotype ofcorresponding cells or tissues in which no insertional event hasoccurred may be produced.

The effect of a transposition event on the phenotype of the transgenicembryo will, of course, depend to some extent on the developmental stageat which transposition occurs. Where transposition is induced, forexample at the single zygote stage, all cells of the embryo developedtherefrom will be homogeneous for the insertional event. Thus, if thetransposition event, e.g. insertional event, results in change inphenotype which is lethal to each cell, the embryo will not develop.Where the transposition event(s) has been induced at a later stage ofdevelopment, each insertional event will be present in the cluster ofcells or tissues derived from cell divisions of the cell in which thetransposition event(s) occurred. This may therefore result in each ofthose cells displaying the same phenotypic variation. For example, ifthe transposition event has been induced in a cell from which some orall cells of a particular tissue of a particular organ is derived, thephenotypic consequences of the insertional event may be limited to thecells, the particular tissue or the particular organ. Where thetransposition event has been induced in a cell from which only some ofthe cells of a particular tissue of a particular organ are derived, amilder phenotype may result from the transposition event than might beobserved if all cells of the tissue display the transposition event.Where the transposition event is lethal to a cell, the cells will notsurvive. If the insertional event is present in all cells from which aparticular tissue or organ is composed, that tissue or organ may notfunction or develop and the embryo may not be viable. Alternatively, thetransposition event may have non-lethal phenotypic consequences. Forexample, the transposition event may have the effect of modulating thefunction of an enzyme in the affected cells, resulting in a relativechange in metabolism compared to the unaffected cells. This maytherefore result in an organ such as the liver, in which a sector oftissue of the variant phenotype is present adjacent to a sector of thesame tissue of the normal phenotype or even a second variant phenotype.The distribution of the variant phenotype within an organism will thusdepend on the stage of embryonic development at which transposition isinduced. Moreover, in some embodiments of the invention induction of a“second round” of transposition may be useful in detecting eitherinversion of the phenotype, caused by excision of an element, or, moreimportantly, modification of the phenotype, caused by a new insertion toan interacting gene.

Phenotypic variations in cells, tissues or organs of the transgenicorganisms may be traced back to transposition events in the genome ofthose cells, tissues or organs.

Accordingly, in a fourth aspect the invention provides a method fordetecting and characterising a genetic mutation in a transgenicorganism, comprising the steps of:

-   -   (a) generating a transgenic embryo and inducing transposition        therein by a method according to the first or third aspect of        the invention or a transgenic organism according to the second        aspect of the invention;    -   (b) identifying in the transgenic embryo or offspring developed        therefrom the presence of a plurality of cells displaying a        variant phenotype;    -   (c) detecting the position of one or more transposon        transposition events in the genome of one or more of the cells;        and correlating the position of the transposition events with        the observed variant phenotype, the position of the        transposition events being indicative of the location of one or        more genetic loci associated with the observed variant        phenotype.

A “transposition event” is a change in genomic sequence caused bytransposon mobilisation and includes insertion events, excision eventsor chromosomal breaks.

Insertion events may be detected by screening for the presence of thetransposon by probing for the nucleic acid sequence of the transposon.Excisions may also be identified by the “signature” sequence left behindupon excision.

A fifth aspect of the invention provides a method for isolating a genewhich is correlated with a phenotypic characteristic in a plurality ofcells in a transgenic animal, comprising the steps of:

-   -   (a) generating a transgenic embryo and inducing transposition        therein by a method according to the first or third aspect of        the invention or a transgenic organism according to the second        aspect of the invention;    -   (b) identifying in the transgenic embryo or offspring developed        therefrom the presence of a plurality of cells displaying the        phenotypic characteristic;    -   (c) detecting the position of one or more transposon        transposition events in the genome of one or more of the cells;        and    -   (d) cloning the genetic loci comprising the insertions.

The locus of the modification may be identified precisely by locatingthe transposon insertion. Sequencing of flanking regions allowsidentification of the locus in databases, potentially without the needto sequence the locus.

In preferred embodiments of the invention, the transposon may be anatural transposon. Preferably, it is a class 2 transposon, such asMinos. Most advantageously, it is Minos. Alternative transposons includemariner, Hermes, piggyBac, hobo and salmonid-type transposons such asSleeping Beauty.

Modified transposons, which incorporate one or more heterologous codingsequences and/or expression control sequences may also be used in theinvention. Such coding sequences may include selectable and/orunselectable marker genes, which may facilitate the identification oftransposons in the genome and cloning of the loci into which thetransposons have been integrated. Suitable markers include fluorescentand/or luminescent polypeptides, such as GFP and derivatives thereof,luciferase, β-galactosidase or chloramphenicol acetyl transferase (CAT).

Such markers may be used in in vivo enhancer or silencer traps and exontraps, by, for example inserting transposons which comprise marker geneswhich are modulated in their expression levels by proximity withenhancers or exons. Constructs for use in exon and enhancer traps aredescribed in EP 0955364. Using the methods of the invention, theplurality of cells or tissues homogeneous for a transposition event maydisplay modulation of expression of marker gene(s), thus enablingefficient trapping of enhancers and/or silencers and/or exons. Moreover,in embodiments where only a proportion of cells or tissues of aparticular type are homogeneous for the transposition event, modulationof the expression of a marker gene may be identified by comparison withcells or tissues of the same type in the same transgenic animal whichdoes not display such modulation.

Accordingly, the invention further provides in a sixth aspect a methodfor isolating an enhancer or a silencer in a transgenic animal,comprising the steps of:

-   -   (a) generating a transgenic embryo and inducing transposition        therein by a method according to the first or third aspect of        the invention or a transgenic organism according to the second        aspect of the invention, wherein the transposon comprises a        reporter gene under the control of a minimal promoter such that        it is expressed at a basal level;    -   (b) assessing the level of expression of the reporter gene in        one or more cells or tissues of the transgenic embryo or        offspring derived therefrom;    -   (c) identifying and cloning genetic loci in one or more of the        cells or tissues in which the modulation of the reporter gene is        increased or decreased compared to the basal expression level;        and    -   (d) characterising the cloned genetic loci in the cell or        tissue.

In a seventh aspect, there is provided a method for isolating an exon ina transgenic animal, comprising the steps of:

-   -   (a) generating a transgenic embryo and inducing transposition        therein by a method according to the first or third aspect of        the invention or a transgenic organism according to the second        aspect of the invention, wherein the transposon comprises a        reporter gene which lack translation initiation sequences but        includes splice acceptor sequences;    -   (b) identifying in the embryo or offspring derived therefrom one        or more cells or tissues in which the reporter gene is        expressed; and    -   (c) cloning the genetic loci comprising the expressed reporter        gene from the cells or tissues.

FIGS. 2, 3 and 4 schematically illustrate gene trap constructs which maybe used in generating embryos for use in these aspects of the invention.

FIG. 2 illustrates a transposase construct which is under the control ofthe tet responsive element (TRE) linked to a minimal promoter and atransposon comprising a marker gene encoding an autofluorescent protein(AFP) under the control of a minimal promoter. In transgenic embryoscomprising both constructs, expression of the transposase may be inducedby activation of the inducible TetO promoter, such that thetransposition of the transposon construct may be achieved. Integrationinto the genome at or near an enhancer site can be detected byexpression of the marker gene.

FIG. 3 illustrates a transposase construct which is under the control ofthe inducible TetO promoter and a transposon comprising an AFPfluorescent reporter gene which lacks translation initiation sequencesbut includes splice acceptor sequences. In transgenic embryos comprisingboth constructs, expression of the transposase may be induced byactivation of the inducible TetO promoter, such that the transpositionof the transposon construct may be achieved. Integration into an intronin the appropriate orientation can be detected by expression of themarker gene.

In a preferred embodiment, transposons may be used to upregulate theexpression of genes. For example, a transposon may be modified toinclude an enhancer or other transcriptional activation element.Mobilisation and insertion of such a transposon in the vicinity of agene upregulates expression of the gene or gene locus. This embodimenthas particular advantage in the isolation of oncogenes, which may beidentified in clonal tumours by localisation of the transposon.

FIG. 4 illustrates a gene activation system which may be used ingenerating embryos for use in this aspect of the invention. FIG. 4illustrates a transposase construct which is under the control of theinducible TetO promoter and a transposon comprising an AFP fluorescentmarker gene similarly under the control of the inducible TetO promoter.In transgenic embryos comprising both constructs, expression of thetransposase may be induced by activation of the inducible TetO promoter,such that the transposition of the transposon construct may be achieved.Further, if the transposed construct comprising the AFP is insertedupstream of an ectopic gene, the gene may be activated and itsphenotypic effect observed upon induction of the TetO promoter.

In conventional methods of inducing genetic modifications bytransposition, in which a mosaic of cells each of which may have uniquetransposition induced genetic modifications is produced, the study ofthe phenotypic results of each transposition event is difficult.Similarly, the study of the effects of natural and artificial stimuli onthe transposed cells is difficult to perform and to interpret. However,in contrast, as the methods of the invention enable the production oftransgenic embryos and organisms in which one or more cluster of cellsor tissues is homogeneous for a single transposition event, the effectof a pharmacological or natural stimulus may be easily observed, e.g. bycomparison of reporter gene expression in a homogeneous cluster of cellswith that in surrounding cells or tissues. Thus, the methods of theinvention may be used to study the response of cells in whichtransposition events have occurred to natural stimuli such asphysiological stimuli, for example, hormones, cytokines and growthfactor or artificial stimuli such as drugs in drug discovery approaches,toxicology studies and the like. Indeed, the methods of the inventionenable the response of a cluster of cells or tissues to a stimulus to beobserved in real time.

Accordingly, in an eighth aspect of the invention, there is provided amethod for identifying a gene responsive to a stimulus in a transgenicanimal comprising the steps of:

-   -   (a) generating a transgenic embryo and inducing transposition        therein by a method according to the first or third aspect of        the invention or a transgenic organism according to the second        aspect of the invention, wherein the transposon comprises a        reporter gene under the control of a minimal promoter such that        it is expressed at a basal level;    -   (b) assessing the level of expression of the reporter gene in        one or more cells or tissues of the of the transgenic embryo or        offspring derived therefrom in the absence of the stimulus;    -   (c) providing the stimulus;    -   (d) identifying and cloning genetic loci in one or more of the        cells or tissues in which the modulation of the reporter gene is        increased or decreased in response to the stimulus compared to        the basal expression level.

The method may further comprise the additional step:

-   -   (e) characterising the cloned genetic loci in the cell or        tissue.

This aspect of the invention may thus be used in the identification ofnovel targets for molecular intervention, including targets for diseasetherapy in humans, plants or animals, development of insecticides,herbicides, antifungal agents and antibacterial agents.

One further application is the discovery of genes responsible forpathogenesis (for example, in mouse disease models). If activation of agene, (e.g. a kinase or a receptor) is involved in the pathogenesis ofthe disease, it is possible that, for example, 50% inactivation of thegene will alleviate or reverse one or more phenotypes of the disease.Therefore, groups of cells with an insertion of a transposon whichinactivates one of the two copies of such a gene will be detectable ashealthy clusters in a diseased background.

The transposon may be inserted into a gene. Preferably, the transposonis inserted into a transcribed gene, resulting in the localisation ofthe transposon in open chromatin. The transposon may be flanked bychromatin opening domain elements, such as locus control regions whichprovide tissue specific expression (Fraser, P. & Grosveld, F. (1998)Curr Opin Cell Biol 10:361-365) or ubiquitously-acting chromatin openingelements—(UCOEs), which enable non-tissue specific expression (forexample see WO 0005393). Other chromatin opening domains which may beused in methods of the invention include CpG rich islands, which maynormally be associated with housekeeping genes or tissue specific genes,or insulators.

Moreover, the transposon may itself comprise, between the transposonends, chromatin opening domains. This will cause activation of thechromatin structure into which the transposon integrates, facilitatingaccess of the inducible transposase in a cell or tissue specific mannerthereto.

Similarly the transposase construct may comprise or be flanked bychromatin opening domain elements.

The ability to regulate the transposition event during embryodevelopment and adult life increases the likelihood of transpositionevents in multiple chromatin domains present in different tissues duringdifferent times of development.

The methods of the invention may advantageously be used in thegeneration of a library of genetically modified organisms, in each ofwhich one or more populations of cells or tissues are homogeneous for agenetic modification produced by transposon mobilisation at apredetermined stage of development. Thus, in a further aspect of theinvention, there is provided a method for producing a library oftransgenic organisms in each of which one or more populations of cellsor tissues are homogeneous for a gene modified by transposonmobilisation, comprising modifying cells by transposon mobilisation bythe method according to a first or second or third aspect of theinvention, wherein step (1) is performed at the predetermined stage ofembryo development.

A library of transgenic organisms produced by such a method forms afurther aspect of the invention.

In one embodiment of the invention, the at least one of the one or moreregulatory sequences which permit expression of the transposase aresequences derived from a sperm specific promoter.

In one embodiment of the invention the nucleotide sequence of the geneencoding the transposase has been mammalianized.

According to the methods of the invention, the activity of thetransposase can be increased or maximized in a target organism bymethods well known in the art including but not limited to optimizingcodon usage to the target organism (for example mouse or rat), asdescribed hereinbelow, increasing the copy number of the transposasegene, or engineering the transposase to increase or maximise activity ina target organism. The activity of a transposase can be increased byprotein engineering methods well known in the art.

In one embodiment of the invention the nucleotide sequence of the geneencoding the transposon has been modified to optimize codon usage.

In one embodiment, the nucleotide sequence of the gene encoding thetransposon has been mammalianized.

In one embodiment of the invention transposition occurs in the sperm,the egg or the early embryo.

In one embodiment of the invention expression of the gene encoding thetransposase is eliminated by gene excision at a lox site by cre or at anFRT site by FLP.

As used herein “one or more copies”, as it refers to a transposon or atransposase of the invention, means 1-10, preferably 1-20, morepreferably 1-50 and most preferably 1-100 or more, ((for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500 or more). In one embodiment the “one or more copies” of thetransposon are integrated in tandem at one or more sites, as definedherein, in the genome (i.e. on different chromosomes).

In one embodiment of the invention, the first adult transgenic organismand/or the progeny comprises between 1 and 100 copies of the transposon.

In one embodiment of the invention the second adult transgenic organismand/or the progeny comprises between 1 and 100 copies of saidtransposase.

In one embodiment the one or more copies of a transposon are integratedat one or more sites in the genome of the first adult organism and/orthe progeny.

In one embodiment the nucleotide sequence of the gene encoding thetransposase is modified to increase transposase activity in saidprogeny. As used herein, “increase” means the activity is at least 5% ormore greater than (for example, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90 or 100% greater than) the activity of an unmodifiedtransposase.

As used herein, “modified or engineered” refers to altering thepolynucleotide or amino acid sequence, for example, of a transposase ofthe invention. The polynucleotide encoding a transposase can comprisecoding sequences for a naturally occurring transposase or can encode analtered transposase with increased activity, as defined herein, ascompared to a naturally occurring transposase. In one embodiment, apolynucleotide sequence encoding a transposase is modified byintroducing one or more mutations. A mutation or mutations in atransposase polynucleotide can be made by site directed mutagenesisusing conventional techniques. A library of mutant polynucleotidescomprising single, double, or higher mutations, can also be preparedusing random mutagenesis techniques. Mutagenesis techniques aredescribed generally, e.g., in Current Protocols in Molecular Biology,Ausubel, F. et al. eds., John Wiley (1998), and random mutagenesis (alsoreferred to as “DNA shuffling”) is the subject of U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458 to Stemmer etal.

As used herein “mutation” refers to a variation in the nucleotidesequence of a gene or regulatory sequence as compared to the naturallyoccurring or normal nucleotide sequence. A mutation may result from thedeletion, insertion or substitution of more than one nucleotide (e.g.,2, 3, 4, or more nucleotides) or a single nucleotide change such as adeletion, insertion or substitution. The term “mutation” alsoencompasses chromosomal rearrangements.

As used herein, “alteration” refers to a change in either a nucleotideor amino acid sequence, as compared to the naturally occurring sequence,resulting from a deletion, an insertion or addition, or a substitution.

As used herein, “deletion” refers to a change in either nucleotide oramino acid sequence wherein one or more nucleotides or amino acidresidues, respectively, are absent.

As used herein, “insertion” or “addition” refers to a change in eithernucleotide or amino acid sequence wherein one or more nucleotides oramino acid residues, respectively, have been added.

As used herein, “substitution” refers to a replacement of one or morenucleotides or amino acids by different nucleotides or amino acidresidues, respectively.

A polynucleotide comprising mutations of a transposase of the inventioncan also be synthesized in a laboratory. Alternatively the transposasemay comprise one or more insertions, substitutions or deletions of aminoacids to provide enhanced activity in the host organism.

In one embodiment, a transposase polynucleotide of the invention isengineered such that the codons are systematically replaced by codonspreferred by the target organism. For example, the coding sequence of atransposase, or a portion thereof, can be analyzed for its codon usage.This codon usage can then be compared with the frequency of codon usagein abundant proteins found in a target organism. The codons of atransposase which have low or zero frequency of use in a target organismcan be modified by, for example, site directed mutagenesis or apolynucleotide can be synthesized in the laboratory. The codonmodifications are made to conform with the codons used in the genes forthe abundantly expressed target organism proteins. Further, segments ofcodons with poly-A signal sequences can be modified to other codons forthe same amino acids. Further, cryptic signal sequences, intron splicesites, and potential methylation sites can be modified. A transposasepolynucleotide sequence which as had at least 1 or more (for example 1,2, 3, 4, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 300 or more) codonsmodified to target organism preferred codons is said to be optimized forcodon usage in a target organism. In one embodiment, optimization forcodon usage in a target organism further comprises modification ofcodons encoding possible signal sequences, intron splice sites, andmethylation sites.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates schematically, the use of a self-inactivatingautonomous transposon construct in an embodiment of the invention. Afirst transgenic organism (A), the genome of which comprises a regulatorconstruct, is crossed with a second transgenic organism (B), the genomeof which comprises a transposon comprising a gene of interest. One ofthe inverted repeats of the transposon is positioned in an intron whichinterrupts the transposase gene. On inducing mobilisation of thetransposon in the progeny of the cross (C) the transposase gene isdisrupted, resulting in stabilised transposition of the gene of interestwith no further transposition events, even in the presence of inducer.

FIG. 2 shows schematically a transposase encoding construct in which thetransposase gene is under the control of the inducible TetO promoter anda transposon comprising an AFP fluorescent marker gene under the controlof a minimal promoter for use in enhancer trap methods of the invention.

FIG. 3 illustrates a transposase construct which is under the control ofthe inducible TetO promoter and a transposon comprising an AFPfluorescent reporter gene which lacks translation initiation sequencesbut includes splice acceptor sequences for use in exon trap methods ofthe invention.

FIG. 4 shows schematically a transposase construct and a transposon foruse in gene traps to identify ectopic genes.

FIG. 5 shows the Minos derived vector pMiCMVGFP. Minos inverted terminalrepeats are shown as thick black arrows. White blocks outside thesearrows indicate the sequences flanking the original Minos element in theD. hydei genome. Arrowheads indicate the positions of primers used todetect Minos excisions. Small arrows indicate the direction oftranscription of the GFP and transposase genes. Black bars representfragments used as probes.

FIG. 6 illustrates the structure of the Minos transposase expressioncassette which may be used in an embodiment of the invention asdescribed in Example 4. The 6.5 kb 5′ flanking region and promoter ofthe ZP3 gene were joined to Minos transposase cDNA (ILMi) and the secondintron and polyadenylation site of the human β globin gene. The relevantrestriction sites are indicated.

FIG. 7 illustrates detection of the Minos transposase transcription byRT-PCR analysis. The different tissues that were analysed are indicated.The murine hypoxantine phosphoribosyltransferase (HPRT) was used as aninternal control. pUC 18 DNA MspI digested was used as a marker (M) andH₂O in the negative control.

FIG. 8 illustrates Southern blot analysis of different offspring showingtransposition events. All DNA samples were BglII digested and probedwith a ³²P labelled GFP probe. Lane 1 is a control (female mousecarrying the GFP transposon as a multicopy on Chr 14 and expressingMinos transposase in developing oocytes). Lanes 2, 5, 6, 7, 8, 10 and 11correspond to progeny of a double transgenic female and wt male. Theyall represent different transposition events. Lanes 2 and 5: mice with 2transposition events. Lanes 3 and 4 correspond to the offspring of themouse in lane 2 with 2 transposition events, showing segregation of theinsertions (lane 3 on chromosome 2, lane 4 on chromosome 14 near thecentromere). Lane 9: Offspring of the mouse shown in lane 8, showingsegregation.

FIG. 9 illustrates the sequence of the parental and four different Minosinsertions in the Mus musculus genome. Chromosomal sequences flankingthe new inserted transposon are represented by capital letters,transposon sequence in small letters and the target site duplication inred. The chromosomal locations of insertions and scaffold numbers fromthe Celera database are indicated.

FIG. 10 illustrates FISH analysis of Minos transposition events.Chromosomes were stained with 4′, 6-diamino-2-phenylindole and probedwith a GFP probe as described in Example 4 Materials and Methods. PanelA: mouse 8218-01 with two transposition events (on chromosome 2 and atthe centromere of chromosome 14; see FIG. 8 lane 2). Red colour(indicated by arrow head): staining after tyramide amplification (seeExample 4 Materials and Methods). Panel B: mouse 8218-02 with twotransposition events (one on chromosome 18 and one near the telomere ofchromosome 14 close to the initial position of the transposon concatemer(FIG. 8 lane 5). Green colour (indicated by arrow head): FITC staining.Yellow arrows indicate transposition events.

FIG. 11 illustrates knock-in (A) and regular constructs (B) for specificexpression of transposase in sperm or egg or for ubiquitous expression.In FIG. 11B, the beta globin 3′ second exon (red), intervening sequences(green) and third exon (red) are added.

FIG. 12 illustrates a trap construct for transgenic mice and/or cloninginto retroviral plasmid.

FIG. 13 is a diagram showing in vivo transposition occurring in the eggor early embryo. Double positive females are obtained by crossing atransposase-positive female with a transposon-positive male or bycrossing a transposase-positive male with a transposon-positive female.

FIG. 14 is a diagram showing in vivo transposition occurring in thesperm. Double positive males are obtained by crossing atransposase-positive female with a transposon-positive male or bycrossing a transposase-positive male with a transposon-positive female.

FIG. 15 is a diagram showing in vivo transposition with the male orfemale contributing the transposon, and the male or female contributingthe transposase. Transposition takes place in the fertilised egg orembryo or the sperm.

FIG. 16 is a diagram depicting generation of a double positivetransposon/transposase transgenic male.

FIG. 17 is a diagram showing mousified Minos transposase knocked intoH1t.

FIG. 18 depicts one embodiment of a breeding scheme according to theinvention. According to this embodiment, the male contributes both thetransposon and the transposase.

FIG. 19 is a Southern blot showing transposition events.

FIG. 20 is a Southern blot showing transposition events.

FIG. 21 shows the results of FISH analysis of transposition events.

FIG. 22 shows the results of FISH analysis of transposition events.

DETAILED DESCRIPTION OF THE INVENTION

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., ShortProtocols in Molecular Biology (1999) 4^(th) Ed, John Wiley & Sons, Inc.

Transposons

Any transposon may be used in the method of the invention. Preferably,the transposon is type 2 transposon, more preferably selected from thegroup consisting of Minos, mariner, Hermes, piggyBac, and SleepingBeauty. Advantageously, the transposon is Minos. Each transposon isadvantageously employed with its natural cognate transposase, althoughthe use of modified and/or improved transposases, for example, amammalianized transposon, is envisaged. Minos transposons, and theircognate transposase, are described in detail in U.S. Pat. No. 5,840,865and European patent application EP 0955364.

The transposon preferably comprises a nucleic acid sequence encoding aheterologous polypeptide. This sequence will be integrated, togetherwith the transposon, into the genome of the cell on transposonintegration. Moreover, it will be excised, together with the transposon,when the latter excises on remobilisation. In a preferred embodiment,the heterologous polypeptide is a selectable marker. This allows cellshaving integrated transposons to be identified and the site ofintegration to be accurately mapped.

Marker Genes

Preferred marker genes include genes which encode fluorescentpolypeptides. For example, green fluorescent proteins (“GFPs”) ofcnidarians, which act as their energy-transfer acceptors inbioluminescence, can be used in the invention. A green fluorescentprotein, as used herein, is a protein that fluoresces green light, and ablue fluorescent protein is a protein that fluoresces blue light. GFPshave been isolated from the Pacific Northwest jellyfish, AequoreaVictoria, from the sea pansy, Renilla reniformis, and from Phialidiumgregarium. (Ward et al. (1982) Photochem Photobiol 35:803-808; Levine etal. (1982) Comp Biochem Physiol 72B:77-85). Fluorescent proteins havealso been isolated recently from Anthoza species (accession nos.AF168419, AF168420, AF168421, AF168422, AF168423 and AF168424).

A variety of Aequorea-related GFPs having useful excitation and emissionspectra have been engineered by modifying the amino acid sequence of anaturally occurring GFP from Aequorea Victoria (Prasher et al. (1992)Gene 111:229-233; Heim et al. (1994) Proc Natl Acad Sci USA91:12501-12504; PCT/US95/14692). Aequorea-related fluorescent proteinsinclude, for example, wild-type (native) Aequorea Victoria GFP, whosenucleotide and deduced amino acid sequences are presented in GenbankAccession Nos. L29345, M62654, M62653 and others Aequorea-relatedengineered versions of Green Fluorescent Protein, of which some arelisted above. Several of these, i.e., P4, P4-3, W7 and W2 fluoresce at adistinctly shorter wavelength than wild type.

Examples of other marker genes which may be used include selectablemarker genes such as genes encoding neomycin, puromycin or hygromycin orcounter-selection genes such as the genes for cytosine deaminase ornitroreductase.

Those skilled in the art are aware of a multitude of marker genes whichmay be used. Any suitable marker gene may be used and it should beappreciated that no particular choice is essential to the presentinvention.

Identification of Insertion and Excision Events

Transposons, and sites from which transposons have been excised, may beidentified by sequence analysis. For example, Minos typically integratesat a TA base pair, and on excision leaves behind a duplication of thetarget TA sequence, flanking the four terminal nucleotides of thetransposon. The presence of this sequence, or related sequences, may bedetected by techniques such as sequencing, PCR and/or hybridisation. Theoriginal insertion site of the transposon is, in one embodiment,additionally marked by Drosophila sequences flanking the transposon.These sites are still present at the original site of the transposonintegration.

Inserted transposons may be identified by similar techniques, forexample using PCR primers complementary to the terminal repeatsequences.

Transposases

Effective transposon mobilisation depends on both efficient delivery ofthe transposable element itself to the host cell and the presence of aneffective cognate transposase in the cell in order to catalysetransposon jumping. A “cognate” transposase, as referred to herein, isany transposase which is effective to activate transposition of thetransposon, including excision of the transposon from a firstintegration site and/or integration of the transposon at a secondintegration site. Preferably, the cognate transposase is the transposasewhich is naturally associated with the transposon in its in vivosituation in nature. However, the invention also encompasses modifiedtransposases, which may have advantageously improved activities withinthe scope of the invention. For example, the sequence of the geneencoding the transposase may be modified to optimise codon usage andthus increase transposition frequencies. In one embodiment, the sequenceis mammalianized. Optimisation of codon usage is a method well known inthe art to increase the expression levels of a given gene. Alternativelythe transposase may comprise one or more insertions, substitutions ordeletions of amino acids to provide enhanced activity in the hostorganism.

The gene encoding the transposase may be provided in the genome of asecond organism which is crossed with a first organism comprising, inits genome, the transposon to produce a double positive embryo for usein the methods of the invention. In an alternative embodiment, one ormore copies of both the transposon and the gene encoding the cognatetransposase are provided in the genome of a first organism, which may becrossed with a second organism comprising one or more copies ofregulatory elements necessary to permit inducible transposase expressionto produce an embryo.

A number of methods are known in the art for introduction of a gene intothe genome of a host cell, and may be employed in the context of thepresent invention. For example, transposase genes may be inserted intothe host cell genome by transgenic techniques. Such methods arediscussed further below.

Regulation of Transposase Expression

Coding sequences encoding the transposase may be operatively linked toregulatory sequences which modulate transposase expression as desired.Control sequences operably linked to sequences encoding the transposaseinclude promoters/enhancers and other expression regulation signals.These control sequences may be selected to be compatible with the hostorganism in which the expression of the transposase is required. Theterm promoter is well known in the art and encompasses nucleic acidregions ranging in size and complexity from minimal promoters topromoters including upstream elements and enhancers.

The promoter is typically selected from promoters which are functionalin cell types homologous to the organism in question, or the genus,family, order, kingdom or other classification to which that organismbelongs, although heterologous promoters may function—e.g. someprokaryotic promoters are functional in eukaryotic cells. The promotermay be derived from promoter sequences of viral or eukaryotic genes. Forexample, it may be a promoter derived from the genome of a cell in whichexpression is to occur. With respect to eukaryotic promoters, they maybe promoters that function in a ubiquitous manner (such as promoters ofα-actin, β-actin, tubulin) or, alternatively, a tissue-specific manner(such as promoters of the genes for pyruvate kinase). In the generationof germline transposition events, the promoters may be derived fromgenes whose expression is induced during gametogenesis, either oogenesisor spermatogenesis. Alternatively, for developmentally regulatedtransposition events such as transposition during zygote development,the promoters may be derived from genes whose expression isdevelopmentally regulated. For expression in the early zygote, promotersfrom maternal effect genes may be used. They may also be promoters thatrespond to specific stimuli, for example promoters that bind steroidhormone receptors. Viral promoters may also be used, for example theMoloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter,the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus(CMV) IE promoter.

According to the invention, the gene encoding the transposase is underthe control of one or more regulatory sequences, meaning that the levelsof expression obtained using e.g. a promoter can be regulated. Forexample the regulatory sequence may be an inducible regulatory sequence.Inducible systems for gene expression are known in the art, and includetetracycline, ecdysone and estrogen-inducible systems or the lacoperator-repressor system.

A widely used system of this kind in mammalian cells is the tetOpromoter-operator, combined with thetetracycline/doxycycline-repressible transcriptional activator tTA, alsocalled Tet-Off gene expression system (Gossen, M. & Bujard, H. (1992)Tight control of gene expression in mammalian cells by tetracyclineresponsive promoters. Proc Natl Acad Sci USA 89:5547-5551), or thedoxycycline-inducible rtTA transcriptional activator, also called Tet-Onsystem (Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W. &Bujard, H. (1995) Transcriptional activation by tetracycline inmammalian cells. Science 268:1766-1769).

In the Tet-Off system, gene expression is turned on when tetracycline(Tc) or doxycycline (Dox; a Tc derivative) is removed from the culturemedium. In contrast, expression is turned on in the Tet-On system by theaddition of Dox. Procedures for establishing cell lines carrying thetranscriptional activator gene and the Tet-regulatable gene stablyintegrated in its chromosomes have been described. For example seehttp://www.clontech.com/techinfo/manuals/PDF/PT3001-1.pdf. For example,the Tet-On system may be employed for tetracycline-inducible expressionof Minos transposase in a transgenic animal.

A doubly transgenic animal may be generated by methods well known in theart including but not limited to transgenesis (microinjection ofoocytes), transfection of ES cells or standard homologous recombinationES cell technology. Both transgenesis and transfection result in randomintegration in the genome but transfection can also be used for ahomologous integration by standard techniques. Two constructs are used:first, a construct containing the rtTA gene under a constitutivepromoter. An example of such construct is the pTet-On plasmid (Clontech)which contains the gene encoding the rtTA activator under control of theCytomegalovirus immediate early (CMV) promoter. The rtTA transcriptionalactivator encoded by this construct is active only in the presence ofDoxycycline. The second construct contains the Minos transposase geneunder control of the tetracycline-response element, or TRE. The TREconsists of seven direct repeats of a 42-bp sequence containing the tetoperator (tetO), and is located just upstream of the minimal CMVpromoter, which lacks the enhancer elements normally associated with theCMV immediate early promoter. Because these enhancer elements aremissing, there is no “leaky” expression of transposase from the TRE inthe absence of binding by rtTA. An example of such construct is thepTRE2 plasmid (Clontech) in the MCS of which is inserted the geneencoding Minos transposase. In cells stably transformed with the twoconstructs, rtTA is expressed but does not activate transcription ofMinos transposase unless Doxycycline is administered to the animal.

Therefore, when, according to a method of the invention, a transgenicanimal comprising both pTet-On and the pTRE2 constructs is crossed withanother animal, the genome of which comprises a transposon, mobilisationof transposons in resulting embryos comprising within their genomes boththe transposon and the gene encoding the transposase will not occur inthe absence of Doxycycline. Administration of Doxycycline thus be usedto induce transposition.

Inducers, e.g. Doxycycline, may be administered to embryos by anysuitable method. In a preferred embodiment of the invention, inducers oftransposase expression are administered via the food or water of theparent organism.

Alternative inducible systems include tamoxifen inducible transposase [amodified oestrogen receptor domain (Indra et al., Nucl Acid Res. 27,4324-27, 1999) coupled to the transposase which retains it in thecytoplasm until tamoxifen is given to the culture], an RU418 inducibletransposase (operating under the same principle with the glucocorticoidreceptor; see Tsujita et al., J Neuroscience, 19, 10318-23, 1999), or anecdysone-inducible system.

The ecdysone-inducible system is based on the heterodimeric ecdysonereceptor of Drosophila, which is induced by the insect hormone, ecdysoneand its derivatives. During metamorphosis of Drosophila melanogaster, acascade of morphological changes is triggered by the steroid hormone20-OH ecdysone, generally referred to as “ecdysone”, via the ecdysonereceptor. Ecdysone responsiveness may be transferred to mammalian cellsby the stable expression of a modified ecdysone receptor that regulatesan optimized ecdysone responsive promoter. Transgenic organisms, e.g.mice expressing the modified ecdysone receptor can activate anintegrated ecdysone responsive promoter upon administration of hormoneor its derivatives e.g. Once the receptor binds ecdysone or muristerone,an analog of ecdysone, the receptor activates the ecdysone-responsivepromoter to give controlled expression of the gene of interest.Ecdysone-based inducible systems are reported to exhibit lower basalactivity and higher inducibility than tetracycline based systems.Further details of ecdysone based inducible systems can be found, forexample, in U.S. Pat. No. 6,245,531 and in No D, Yao T P, Evans R MEcdysone-inducible gene expression in mammalian cells and transgenicmice, Proc Natl Acad Sci USA 1996 April 93:3346-51, the contents of eachof which are herein incorporated by reference.

The lac operator-repressor system has recently been shown to befunctional in mammals, in particular the mouse. Cronin et al, Genes andDevelopment, 15, 1506-1517 (2001), the contents of which are hereinincorporated by reference, describes the use of a lac repressortransgene that resembles a typical mammalian gene both in codon usageand structure and that expresses functional lac repressor proteinubiquitously in mice to control the expression of a reporter gene underthe control of the lac promoter. Expression of the reporter gene isreversible using the lactose analog IPTG provided in the drinking waterof the mouse or mother of the embryo or nursing pup. The lacoperator-repressor system may thus be adapted for use to regulateexpression of the transposase by placing the transposase gene under thecontrol of a lac promoter.

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above.

The use of locus control regions (LCRs) is also envisaged. LCRs arecapable of conferring tightly-regulated tissue specific control ontransgenes, and to greatly increase the fidelity of transgeneexpression. A number of LCRs are known in the art. These include theβ-globin LCR (Grosveld et al., (1987) Cell 51:975-985); α-globin (Hattonet al., (1990) Blood 76:221-227; and CD2 (Festenstein et al., (1996)Science 271:1123-1125) the T cell specific CD4 (Boyer et al J Immunol1997, 159:3383-3390), and TCR loci (Diaz P, et al Immunity 1994,1:207-217; Ortiz et al EMBO J 1997, 16:5037-5045; Hong et al Mol CellBiol 1997, 17:2151-2157.) the B-cell-specific MHC class II Ea (Carson etal Nucleic Acids Res 1993, 21:2065-2072), the macrophage-specificlysozyme gene (Bonifer et al EMBO J 1990, 9:2843-2848), theneuron-specific S100 gene (Friend et al J Neurosci 1992, 12:4337-4346),the liver-specific LAP gene (Talbot et al Nucleic Acids Res 1994,22:756-766), the human growth hormone locus (Jones et al Mol Cell Biol1995, 15:7010-7021), plus immunoglobulins, muscle tissue, and the like.Further details on LCRs are provided in Fraser, P. & Grosveld, F.(1998). Curr. Opin. Cell Biol. 10, 361-365 and Li, Q., Harju, S. &Peterson, K. R. (1999). Trends Genet. 15:403-408.

Alternatively, gene domains that need to be open and switched-on in allcells of the body; i.e. gene domains whose proteins (such as enzymes forgenerating energy from sugars), are needed by all cells for survival andwhich are therefore ubiquitously expressed may be exploited to enableexpression of the transposon and/or transposase in every tissue.Examples of such ubiquitously-acting chromatin opening elements—(UCOEs)include the human genes known as TBP and hnRNPA2. Further details of theuse of such UCOEs may be found in Antoniou, M. and Grosveld, F. (1999).(Genetic approaches to therapy for the haemoglobinopathies. in: BloodCell Biochemistry, Volume 8: Hematopoiesis and Gene Therapy Fairbairnand Testa eds. Kluwer Academic/Plenum Publishers, New York. pp 219-242)and in PCT/GB99/02357 (WO 0005393), the contents of both of which areherein incorporated by reference.

Regulation of transposase and/or transposon expression may also beachieved through the use of ES cells. Using transformed ES cells toconstruct chimeric embryos, it is possible to produce transgenicorganisms which contain the transposase genes or transposon element inonly certain of their tissues. This can provide a further level ofregulation.

Maximising Efficiency of Transposition

As described above and for example, in WO 01/71019 and WO 02/062991,transposition is achieved by the action of the transposase enzyme on theterminal repeat sequences of the integrated transposon, resulting inexcision of the transposon from its original position in the “host”genome and reinsertion of the transposon at a different position in thegenome.

As with most biochemical processes, this process can be made to be moreefficient by simply improving the concentration of substrates, highlevels of the terminal repeats sequences, i.e. an increase in copynumber and high levels of the transposase enzyme.

An increase in copy number can be achieved by generating multiple copyarrays at the original insertion site. For example, 10 to 100 copies canbe generated through standard transgenesis or using a PAC vector.Alternatively, multiple copies can be generated by the presence ofmultiple insertions at different sites in the genome.

The sequence of the transposase may be modified to optimise codon usageand thus, increase transposition frequencies. Optimisation of codonusage is a method well known in the art to increase the expressionlevels of a given gene.

Thus, the efficiency of the fly transposase in mammalian cells oranimals may be increased by increasing its concentration as a result ofa more efficient translation from mRNA by replacing the fly codon usageto mammalian codon usage.

Assays for determining transposase efficiency can include a standardtransposition assay as described, for example, by Klinakis et al.;Insect Molecular Biology, 9 (3), 269-275, 2000.

The concentration of transposase mRNA can also be increased by includingin the transposase mRNA sequence 5′ and 3′ sequences found in abundantstable mRNAs such as those encoding growth hormone, globin, actin oralbumin.

Transgenic Organisms

Methods of the invention may employ one or more transgenic organismshaving integrated in the genome the transposon, a gene encoding thecognate transposase or both.

The introduction of the transposon or gene encoding the transposase maybe accomplished by any available technique, includingtransformation/transfection, delivery by viral or non-viral vectors andmicroinjection. Each of these techniques is known in the art. Thetransposon and the gene encoding the transposase may be inserted usingthe same or different methods. For example, the Drosophila P-element maybe used to introduce a Minos transposase construct into Drosophila.

In a preferred embodiment, the transposon or gene encoding thetransposase may be inserted into the host cell genome by transgenictechniques, for example to produce a transgenic animal comprising atransposon, a gene encoding a cognate transposase or both. Where thetransgenic animal comprises both the transposon and the gene encodingthe transposase, both constructs can be inserted using the same ordifferent methods. Where delivery of the construct is by viral vector, acomposite vector comprising both the transposon and the gene encodingthe transposase under the control of a control sequence such as the Tetoperator may be used. Alternatively, separate vectors may be used.

Any suitable transgenic animal may be used in the present invention.Animals include animals of the phyla cnidaria, ctenophora,platyhelminthes, nematoda, annelida, mollusca, chelicerata, uniramia,crustacea and chordata. Uniramians include the subphylum hexapoda thatincludes insects such as the winged insects. Chordates includevertebrate groups such as mammals, birds, fish, reptiles and amphibians.Particular examples of mammals include non-human primates, cats, dogs,ungulates such as cows, goats, pigs, sheep and horses and rodents suchas mice, rats, gerbils and hamsters.

Techniques for producing transgenic animals which may be used in themethod of the invention are well known in the art. A useful generaltextbook on this subject is Houdebine, Transgenic animals—Generation andUse (Harwood Academic, 1997)—an extensive review of the techniques usedto generate transgenic animals.

In a preferred embodiment, the animal is an insect. Methods forproducing transgenic insects which may be used in the method of theinvention are well known (see for example Loukeris et al.(1995), Science270, 2002-2005). Briefly, a transposable element carrying the gene ofinterest is inserted into a preblastoderm embryo using e.g.microinjection. Preferably, the new genetic material is placed at thepolar plasm, which is the section of egg destined to become the stillnascent insect's own egg or sperm cells. After many divisions of thenuclear material, most of it segregates to the periphery where it willbecome the nuclei of the insect's body. A small number of nuclei migrateto the pole to become egg cells on maturity. If these cells incorporatethe transgene, progeny will be transgenic. Further details of producingtransgenic insects are provided in Loukeris et al (1995), Science 270,2002-2005 and O'Brochta and Atkinson (1998) Scientific American 279,60-65.

In another preferred embodiment, the animal is preferably a mammal.Advances in technologies for embryo micromanipulation now permitintroduction of heterologous DNA into, for example, fertilised mammalianova. For instance, totipotent or pluripotent stem cells can betransformed by microinjection, calcium phosphate mediated precipitation,liposome fusion, retroviral infection or other means, the transformedcells are then introduced into the embryo, and the embryo then developsinto a transgenic animal. In a highly preferred method, developingembryos are infected with a retrovirus containing the desired DNA, andtransgenic animals produced from the infected embryo. In a mostpreferred method, however, the appropriate DNAs are coinjected into thepronucleus or cytoplasm of embryos, preferably at the single cell stage,and the embryos allowed to develop into mature transgenic animals aftertransfer into pseudopregnant recipients. Those techniques are well known(see reviews of standard laboratory procedures for microinjection ofheterologous DNAs into mammalian fertilised ova, including Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Press 1986);Krimpenfort et al., Bio/Technology 9:844 (1991); Palmiter et al., Cell,41:343 (1985); Kraemer et al., Genetic manipulation of the MammalianEmbryo, (Cold Spring Harbor Laboratory Press 1985); Hammer et al.,Nature, 315:680 (1985); Wagner et al., U.S. Pat. No. 5,175,385;Krimpenfort et al., U.S. Pat. No. 5,175,384, the respective contents ofwhich are incorporated herein by reference.).

Transgenic animals may also be produced by nuclear transfer technologyas described in Schnieke, A. E. et al., 1997, Science, 278: 2130 andCibelli, L B. et al., 1998, Science, 280: 1256. Using this method,fibroblasts from donor animals are stably transfected with a plasmidincorporating the coding sequences for a polypeptide of interest underthe control of regulatory sequences. Stable transfectants are then fusedto enucleated oocytes, cultured and transferred into female recipients.

Analysis of animals which may contain transgenic sequences wouldtypically be performed by either PCR or Southern blot analysis followingstandard methods.

By way of a specific example for the construction of transgenic mammals,such as cows, nucleotide constructs comprising a sequence encoding a DNAbinding molecule are microinjected using, for example, the techniquedescribed in U.S. Pat. No. 4,873,191, into oocytes which are obtainedfrom ovaries freshly removed from the mammal. The oocytes are aspiratedfrom the follicles and allowed to settle before fertilisation withthawed frozen sperm capacitated with heparin and prefractionated byPercoll gradient to isolate the motile fraction.

The invention provides for transgenic animals produced by any methodknown in the art. For example, transgenic animals useful according tothe invention are produced by methods including but not limited to 1)microinjection of fertilized eggs; 2) transfection or infection of EScells followed by injection of ES cells into blastocysts, resulting inchimeric offspring, or fusion of ES cells with tetraploid embryosresulting in totally ES derived offspring; and 3) cloning by nucleartransfer. Homologous recombination may occur when using ES cells or whenusing the method of nuclear transfer.

The fertilised oocytes are centrifuged, for example, for eight minutesat 15,000 g to visualise the pronuclei for injection and then culturedfrom the zygote to morula or blastocyst stage in oviducttissue-conditioned medium. This medium is prepared by using luminaltissues scraped from oviducts and diluted in culture medium. The zygotesmust be placed in the culture medium within two hours followingmicroinjection.

Oestrous is then synchronized in the intended recipient mammals, such ascattle, by administering coprostanol. Oestrous is produced within twodays and the embryos are transferred to the recipients 5-7 days afteroestrous. Successful transfer can be evaluated in the offspring bySouthern blot.

Alternatively, the desired constructs can be introduced into embryonicstem cells (ES cells) and the cells cultured to ensure modification bythe transgene. The modified cells are then injected into the blastulaembryonic stage and the blastulas replaced into pseudopregnant hosts.The resulting offspring are chimeric with respect to the ES and hostcells, and nonchimeric strains which exclusively comprise the ES progenycan be obtained using conventional cross-breeding. This technique isdescribed, for example, in WO91/10741.

Alternative methods for delivery and stable integration of transposonsand/or genes encoding transposases into the genome of host animalsinclude the use of viral vectors, such as retroviral vectors, adenoviralvectors, baculoviral vectors and herpesviral vectors. Such techniqueshave moreover been described in the art, for example by Zhang et al.(Nucl. Ac. Res., 1998, 26:3687-3693).

Suitable viral vectors may be retroviral vectors, and may be derivedfrom or may be derivable from any suitable retrovirus. A large number ofdifferent retroviruses have been identified. Examples include: murineleukaemia virus (MLV), human immunodeficiency virus (HIV), simianimmunodeficiency virus, human T-cell leukaemia virus (HTLV), Equineinfectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV). A detailed list of retroviruses may befound in Coffin et al., 1997, “retroviruses”, Cold Spring HarbourLaboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763.

Details on the genomic structure of some retroviruses may be found inthe art. By way of example, details on HIV and Mo-MLV may be found fromthe NCBI GenBank (Genome Accession Nos. AF033819 and AF033811,respectively).

Retroviruses may be broadly divided into two categories: namely,“simple” and “complex”. Retroviruses may even be further divided intoseven groups. Five of these groups represent retroviruses with oncogenicpotential. The remaining two groups are the lentiviruses and thespumaviruses. A review of these retroviruses is presented in Coffin etal., 1997 (ibid).

Host range and tissue tropism varies between different retroviruses. Insome cases, this specificity may restrict the transduction potential ofa recombinant retroviral vector. For this reason, many gene therapyexperiments have used MLV. A particular MLV that has an envelope proteincalled 4070A is known as an amphotropic virus, and this can also infecthuman cells because its envelope protein “docks” with a phosphatetransport protein that is conserved between man and mouse. Thistransporter is ubiquitous and so these viruses are capable of infectingmany cell types.

Replication-defective retroviral vectors are typically propagated, forexample to prepare suitable titres of the retroviral vector forsubsequent transduction, by using a combination of a packaging or helpercell line and the recombinant vector. That is to say, that the threepackaging proteins can be provided in trans.

A “packaging cell line” contains one or more of the retroviral gag, poland env genes. The packaging cell line produces the proteins requiredfor packaging retroviral DNA but it cannot bring about encapsidation dueto the lack of a psi region. The helper proteins can package apsi-positive recombinant vector to produce the recombinant virus stock.This virus stock can be used to transduce cells to introduce the vectorinto the genome of the target cells. A summary of the availablepackaging lines is presented in Coffin et al., 1997 (ibid).

The lentivirus group can be divided into “primate” and “non-primate”.Examples of primate lentiviruses include human immunodeficiency virus(HIV), and simian immunodeficiency virus (SIV). The non-primatelentiviral group includes the prototype “slow virus” visna/maedi virus(VMV), as well as the related caprine arthritis-encephalitis virus(CAEV), equine infectious anaemia virus (EIAV) and the more recentlydescribed feline immunodeficiency virus (FIV) and bovineimmunodeficiency virus (BIV). See, for example, Rovira et al., (2000)Blood 96:4111-4117; Reiser et al., (2000) J. Virol. 74:10589-99; Mulder,M. P et al. (1995), Hum Genet 96:133-141):10589; Lai et al., Proc NatlAcad Sci USA (2000) 97:11297-302; Southern, E. M. (1975), J. Mol. Biol98; 503-517; and Saulnier et al., (2000) J Gene Med 2:317-25.

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells. In contrast, other retroviruses—such asMLV—are unable to infect non-dividing cells such as those that make up,for example, muscle, brain, lung and liver tissue.

A number of vectors have been developed based on various members of thelentivirus sub-family of the retroviridae and a number of these are thesubject of patent applications (WO-A-98/18815; WO-A-97/12622). Preferredlentiviral vectors are based on HIV, SIV or EIAV. The simplest vectorsconstructed from HIV-1 have the complete HIV genome except for adeletion of part of the env coding region or replacement of the nefcoding region. Notably these vectors express gag/pol and all of theaccessory genes and hence require only an envelope to produce infectiousvirus particles. Of the accessory genes vif, vpr, vpu and nef arenon-essential.

One preferred general format for HIV-based lentiviral vectors is, HIV5′LTR and leader, some gag coding region sequences (to supply packagingfunctions), a reporter cassette, the rev response element (RRE) and the3′LTR. In these vectors gag/pol, accessory gene products and envelopefunctions are supplied either from a single plasmid or from two or moreco-transfected plasmids, or by co-infection of vector containing cellswith HIV.

The adenoviral vector system is also useful according to the methods ofthe invention. The adenovirus is a double-stranded, linear DNA virusthat does not go through an RNA intermediate. There are over 50different human serotypes of adenovirus divided into 6 subgroups basedon the genetic sequence homology all of which exhibit comparable geneticorganisation. Human adenovirus group C serotypes 2 and 5 (with 95%sequence homology) are most commonly used in adenoviral vector systemsand are normally associated with upper respiratory tract infections inthe young.

Adenoviruses/adenoviral vectors which may be used in the invention maybe of human or animal origin. As regards the adenoviruses of humanorigin, preferred adenoviruses are those classified in group C, inparticular the adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12(Ad12). Among the various adenoviruses of animal origin, canineadenovirus, mouse adenovirus or an avian adenovirus such as CELO virus(Cotton et al., 1993, J Virol 67:3777-3785) maybe used. HSV vectors maybe derived from, for example, HSV1 or HSV2 strains, or derivativesthereof. Attenuated strains may be used for example strain 1716 (MacLeanet al., 1991, J Gen Virol 72: 632-639), strains R3616 and R4009 (Chouand Roizman, 1992, PNAS 89:3266-3270) and R930 (Chou et al., 1994, J.Virol 68: 8304-8311) all of which have mutations in ICP34.5, and d27-1(Rice and Knipe, 1990, J. Virol 64:1704-1715) which has a deletion inICP27. Alternatively strains deleted for ICP4, ICP0, ICP22, ICP6, ICP47,vhs or gH, with an inactivating mutation in VMW65, or with anycombination of the above may also be used to produce HSV strains of theinvention.

The terminology used in describing the various HSV genes is as found inCoffin and Latchman, 1996. Herpes simplex virus-based vectors. In:Latchman DS (ed). Genetic manipulation of the nervous system. AcademicPress: London, pp 99-114.

Baculovirus vectors may moreover be employed in the invention. Thebaculovirus Autographa californica multiple nuclear polyhedrosis virus(AcMNPV) is a DNA virus which can replicate only in cells of certainlepidopteran insects and has been used widely for expression ofrecombinant proteins in insect cells. Baculoviruses such as AcMNPV havebeen used recently for introducing heterologous DNA with high efficiencyin a variety of mammalian cells, such as a hepatoma cell line andprimary liver cells, and endothelial cells (Boyce F M, Bucher N L (1996)Baculovirus-mediated gene transfer into mammalian cells. Proc Natl AcadSci USA 93, 2348-52; Airenne K J, Hiltunen M O, Turunen M P, Turunen AM, Laitinen O H, Kulomaa M S, Yla-Herttuala S (2000)Baculovirus-mediated periadventitial gene transfer to rabbit carotidartery. Gene Ther 7, 1499-1504). Moreover, baculovirus vectors for genetransfer, methods for introducing heterologous DNA into their genome andprocedures for recombinant virus production in insect cell cultures areavailable commercially; furthermore, baculoviruses cannot normallyreplicate in mammalian cells, so there is no need to engineer them forthis use.

Construction of vectors for use in methods of the invention may employconventional ligation techniques. Isolated viral vectors, plasmids orDNA fragments are cleaved, tailored, and religated in the form desiredto generate the plasmids required. If desired, analysis to confirmcorrect sequences in the constructed vectors is performed in a knownfashion. Transposon presence and/or mobilisation may be measured in acell directly, for example, by conventional Southern blotting, dotblotting, PCR or in situ hybridisation, using an appropriately labelledprobe which may be based on a sequence present in the transposon. Thoseskilled in the art will readily envisage how these methods may bemodified, if desired. Vectors useful in the present invention areadvantageously provided with marker genes to facilitate transposonidentification and localisation as described above.

Uses of the Invention

The methods of the present invention enables the generation oftransgenic embryos and organisms comprising one or more clonalpopulations of cells homogeneous for one or more individual mutations.Thus, transgenic embryos and animals can be produced in which a clusterof cells, a tissue or tissues, or an organ or group of organs each sharethe same genetic modification. The presence of the same geneticmodification in a number of cells, tissues or organs enables convenientphenotypic and genotypic analysis of the modification and moreoverenables the comparison of the effects of a particular gene modificationto be compared with corresponding wild-type genes or indeed other genemodifications in the same type of cell, tissue or organ within the sameorganism.

The invention may further be used to monitor gene expression patterns ofmodified genes during embryo development and in adult cells and tissues.

Transposons, and sites from which transposons have been excised, may beidentified by sequence analysis. For example, Minos typically integratesat a TA base pair, and on excision leaves behind a footprint, consistingof duplication of the target TA sequence, flanking the four terminalnucleotides of the transposon. The presence of the sequence of Minos,its footprint, or related sequences, may be detected by techniques suchas sequencing, PCR or hybridisation.

Inserted transposons may be identified by similar techniques, forexample using PCR primers complementary to the terminal repeatsequences.

The invention allows functional mapping of a genome by permittingprecise gene modulation at predetermined stages of development andsubsequent detection using transposons. Thus, the invention provides forefficient intracellular transposon mobilisation and insertion into thecell genome of one or more cells or tissues of an organism, providingexon trapping functionality in cells of transgenic organisms at earlystages of development. The induction of transposon mobilisation at suchstages of embryonic development enables the generation of clusters ofcells or tissues which are homogeneous for a transposed gene. This maytherefore enable rapid and efficient detection of a change in phenotypeand/or identification of the modified gene.

An example of a suitable trap construct is given in FIG. 12.

The invention, in an advantageous embodiment, allows genes to be markedfor functional genetic analysis in a group of cells or tissues, orknocked out, by transposon insertion and then specifically identifiedthrough the transposon “tag” without requiring costly and time-consuminggenetic analyses, and frequently without significant amounts ofsequencing.

A further embodiment of the invention provides for the generation oflibraries of transgenic organisms, such as transgenic mice. Target genesmay be identified phenotypically according to the phenotype of one ormore cells, tissues or organs, and identified genetically bydetermination of the transposon insertion site. Inducible expressionsystems, as described above, may be used to regulate the switch betweenpartial and antisense-induced complete knockout of a gene. Somatic cellscarrying transposon insertions can be immortalized, for example byderiving immortal cell lines by standard methodologies, or by generatingtransgenic animal lines by nuclear implantation methodologies.

Such libraries can be used for phenotypic analysis and identification ofgene associations. The present methods allow advantages over the currentmethods.

In inherited diseases such as the haemoglobinopathies, haemophilia,cystic fibrosis, and muscular dystrophy, well defined mutations insingle genes or their regulatory elements result in inappropriate geneexpression with clinical consequences which profoundly effect the lifestyle and life expectancy of affected individuals.

However in other diseases particularly those related to aging, such asthe dementias; psychiatric disorders such as schizophrenia and manicdepression; bone and joint related inflammatory conditions; obesity,insulin resistance, type 2 diabetes and related vascular andcardiovascular conditions; the genetic component is more complex andstill poorly understood (see Lander and Schork, Science (1994) 265,2037-2048).

Where multiple mutations in different targets determine factors such asthe time of disease onset (eg post menopausal insulin resistance) andseverity of the disease state (eg pre-disposition to vascular diseaseand stroke) strategies based on random alkylation of inbreed mousestrains, and transgenic mice developed from single gene deletions invast ES cell libraries seem destined to fail.

The present invention provides a more attractive strategy through therapid generation of randomly “tagged” mouse strains starting with abackground known to be prone to a disease state, and therefore likely tohave existing mutations in interrelated disease causing genes. Thebackground can be selected by using founder cell lines carrying multiple“dormant” transposons at known integration sites. Regulatabletransposase activity will allow the rapid generation of mouse librariesin different genetic backgrounds reflecting differing models of disease.

For example it has been shown that the mutations in the insulin receptorcause mild to severe hyperinsulinemia dependent on the mouse geneticbackground (see Kido (2000) Diabetes 49, 589-596) indicating theinvolvement of other genes in the predisposition to insulin resistance.Thus generation of a tagged mouse library using in vivo genetransposition technology in the “mild phenotype” mouse background shouldlead to selection of animals with a more severe phenotype. Sequenceanalysis of DNA flanking new transposition events will then identify newcandidate disease causing genes which contribute to the onset of thesevere phenotype.

Candidate disease genes can then become the focus of further studies todetermine their precise role in animal models, and validation of adisease related role in man.

Target validation in man will utilise existing clinical and geneticdatabases, containing DNA and clinical information on relevant patientand control groups.

Phenotypic analysis of the transgenic organisms created can be throughsimple and rapid measurements including changes in a metabolite, protein(e.g. insulin), lipid or carbohydrate (e.g. when measuring glucosetolerance) present in urine, blood, spinal fluid or tissue.

Other phenotypic characteristics can be analysed by measuringbehavioural patterns or responses to external stimuli by using testssuch as light, sound, memory and stress tests.

Other measurable phenotypic characteristics include growth and ageingparameters, tumour growth, obesity and so forth which can be measured byassessing, for example, weight, fat content and growth rate. Furthermorechanges in other measurable features such as blood pressure, heart rate,lung function and so forth can be assessed.

Advantageously, the transposon technology of the present inventionallows the production of libraries of transgenic animals without therequirement for extensive storage. Previous methods of generatingtransgenic animals involve methods such as chemical mutagenesis in whichmutations are generated. These methods involve multiple mutations peranimal (e.g. mouse). The animals, once generated are analysedphenotypically and then need to be archived/stored for use in thefuture. The present invention allows the generation of starter celllines or starter transgenic organisms having different transposonsinserted into the genome. These cell lines or organisms can then be usedby breeding with transgenic animals carrying a transposase to generate anew library.

In another embodiment, the methods of the invention may be used togenerate libraries of ES cells with different transposon insertionsdistributed throughout the genome. These can be sequenced andcharacterised. The ES cells can conveniently be stored for future use.

In an alternative embodiment, the methods of the invention may be usedto “mark” genes whose expression is modulated by external stimuli. Thus,an embryo, organism, or tissue or cell derived from either, which hasbeen exposed to transposon mobilisation with a marked transposon issubjected to treatment with an external stimulus, such as a candidatedrug or other test agent, and modulation of the expression of the markerobserved. Cells in which the marker is over or under-expressed arelikely to have the transposon inserted in or near a gene which isupregulated or downregulated in response to the stimulus. The inventionmay thus be used to provide in vivo enhancer trap and exon trapfunctions, by inserting transposons which comprise marker genes whichare modulated in their expression levels by the proximity with enhancersor exons.

This approach is useful for the study of gene modulation by drugs indrug discovery approaches, toxicology studies and the like. Moreover, itis applicable to study of gene modulation in response to naturalstimuli, such as hormones, cytokines and growth factors, and theidentification of novel targets for molecular intervention, includingtargets for disease therapy in humans, plants or animals, development ofinsecticides, herbicides, antifungal agents and antibacterial agents.

The invention is further described, for the purpose of illustration, inthe following examples.

EXAMPLES A: Activation of Minos In Vivo Using Doxycycline Example 1Generation of Transposon Carrying Mice and Transposase Carrying Mice

Two transgenic mouse lines are generated. The transposon-carrying line(line MCG) contains a tandem array of a fragment containing a Minostransposon containing the GFP gene under the control of thecytomegalovirus promoter. The transposon is engineered such that almostall sequence internal to the inverted repeats is replaced by the CMV/GFPcassette. Not containing the transposase-encoding gene, this transposonis non-autonomous, and can only be mobilized when a source oftransposase is present. The second transgenic mouse line contains theMinos transposase gene expressed under the control of the induciblepromoter.

Transposon MiCMVGFP was constructed as follows: The plasmid pMILRTetR(Klinakis et al. (2000) Ins. Mol. Biol. 9, 269-275 (2000b) was cut withBamHI and religated to remove the tetracycline resistance gene betweenthe Minos ends, resulting in plasmid pMILRΔBamHl. An Asp718/SacIfragment from pMILRΔBam Hi, containing the Minos inverted repeats andoriginal flanking sequences from D. hydei, was cloned into plasmidpPolyIII-I-lox (created by insertion of the loxP oligo:

ATAACTTCGTATAGCATACATTATACGAAGTTATinto the Asp71 8 site of the vector pPolyIII-I (accession No. M18131)),resulting in plasmid ppolyMILRΔBamH. The final construct (pMiCMVGFP,FIG. 5) used for the generation of transgenic mice, was created byinserting into the Spe I site of ppolyMILRΔBamHI the 2.2 kb SpeIfragment from plasmid pBluescriptGFP, containing a humanised GFP gene(from Clontech plasmid pHGFP-S65T) driven by the CMV promoter andfollowed by the SV40 intervening sequence and polyadenylation signal.

The transposon-carrying MCG line was constructed by microinjecting the3.2 kb XhoI fragment from the pMiCMVGFP plasmid into FVB×FVB fertilizedoocytes. Transgenic animals were identified by Southern blotting of DNAfrom tail biopsies, using GFP DNA as a probe.

The transposase-expressing line is generated via methods known in theart and described herein, including but not limited to microinjection offertilized eggs; transfection or infection of ES cells followed byinjection of ES cells into blastocysts or fusion of ES cells withtetraploid embryos, or alternatively, cloning by nuclear transfer. Thetransposase gene may be introduced into embryonic stem cells viastandard transfection or homologous recombination ES cell technology.The ES cells are injected into blastocysts to obtain transgenic animalsvia standard procedures (Manipulating the mouse embryo, Hogan et al.,Cold Spring Harbor Press, 1994). Two constructs are used: First, aconstruct containing the rtTA gene under a constitutive promoterexpressed in the target cells. The construct used is the pTet-On plasmid(Clontech) which contains the gene encoding the rtTA activator undercontrol of the Cytomegalovirus immediate early (CMV) promoter. The rtTAtranscriptional activator encoded by this construct is active only inthe presence of Doxycycline. The second construct contains the Minostransposase gene under control of the tetracycline-response element, orTRE. The TRE consists of seven direct repeats of a 42-bp sequencecontaining the tet operator (tetO), and is located just upstream of theminimal CMV promoter, which lacks the enhancer elements normallyassociated with the CMV immediate early promoter. Because these enhancerelements are missing, there is no “leaky” expression of transposase fromthe TRE in the absence of binding by rtTA. The second construct used isthe pTRE2 plasmid (Clontech) in the multiple cloning site (MCS) of whichis inserted the gene encoding Minos transposase. In cells stablytransformed with the two constructs, rtTA is expressed but does notactivate transcription of Minos transposase unless Doxycycline isadministered.

Transgenic animals are identified by Southern blotting of DNA from tailbiopsies, using a transposase cDNA fragment as a probe.

Example 2 Activation of Minos In Vivo

A transgenic mouse of the transposon-carrying MCG line is crossed with atransgenic mouse of the transposase carrying line. Mobilisation oftransposons in resulting embryos comprising within their genomes boththe transposon and the gene encoding the transposase will only occur inthe presence of Doxycycline. Doxycycline is administered to the embryosin the water of the maternal organism. Doxycycline is only administeredfor a limited amount of time (one day-day 2 of gestation) in order torestrict the potential number of transposition events.

On birth, the transgenic offspring developed from the embryos areisolated and various cells and tissues are used for genotyping.

Example 3 Detection of Transposition

A PCR assay for transposon excision is used to detect activetransposition by Minos transposase in the mouse tissues, using primersthat hybridise to the non-mobile Drosophila hydei sequences which flankthe Minos transposon in the constructs (Klinakis et al. (2000) Ins. Mol.Biol. 9, 269-275). In Drosophila cells, transposase-mediated excision ofMinos is followed by repair of the chromatid which usually leaves acharacteristic 6-base pair footprint (Arca et al. (1997) Genetics 145,267-279). With the specific pair of primers used in the PCR assay thiscreates a diagnostic 167 bp PCR fragment (Catteruccia F. et al. (2000)Proc. Natl. Acad. Sci. USA 97, 2157-2162).

Genomic DNA from different tissues is isolated with the DNeasyTissue-Kit (QIAGEN) according to the manufacturers instructions. PCRreactions are performed using primers 11DML:

(5′AAGTGTAAGTGCTTGAAATGC-3′)

and GOUM67:

(5′-GCATCAAATTGAGTTTTGCTC-3′).

PCR conditions are as follows: 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5ruM MgCl₂, 0.001% gelatin; 1.2 units Taq 2000™ DNA Polymerase(STRATAGENE), 200 g template DNA and 10 pmol of each primer per 25 μlreaction. 43 or 60 cycles of 30″ at 94° C., 30″ at 59° C. and 30″ at 72°C. were performed. PCR products are cloned into the PCRII TA cloningvector (Invitrogen) and are sequenced using the T7 primer.

The diagnostic band is present in certain tissues of the transgenicoffspring. The identity of the fragment is confirmed by Southern blotanalysis using a labelled DNA probe specific for the amplified sequence(data not shown). Clusters of cells are shown to be homogeneous for thesame transposed gene.

B: Activation of Minos In Vivo Using Transposase Under the Control ofthe ZP3 Promoter Example 4

In this example, it is demonstrated that, by placing expression of thegene encoding the transposase under the control of a gene regulatorysignal produced at a particular stage of development, transposition canbe induced at that stage of embryo development. In this example, thegene encoding the transposase was placed under the control of the ZP3promoter, and so the transposase was only expressed in growing oocytesduring a 2-to-3-week period of oogenesis. The Minos transposaseexpressed in growing oocytes catalysed the excision of a modified, nonautonomous Minos transposon and promoted its re-integration into newsites of the genome.

FIG. 11 shows alternative constructs (constructs for knock-in and forregular transgenesis) in which the transposase is inserted in theendogenous sperm specific H1t gene for transposition in sperm.Alternative constructs are made for egg specific expression orubiquitous expression by replacing H1t sequences with equivalentsequences flanking the start of the Zp3 (egg specifically expressed) orhnRNP (ubiquitously expressed) gene. The invention encompasses any eggspecific, sperm specific or endogenous regulatory sequence known in theart.

C: Mammalian Codon Usage in Minos Sequence Example 5 ImprovingTransposase

One of the ways to improve the efficiency of the fly transposase inmammalian cells or animals is to increase its concentration as a resultof a more efficient translation from mRNA by replacing the fly codonusage to mammalian codon usage. To this end we replaced the codingsequence of the fly Minos transposase with the sequence:

-   -   SEQ New: 1026 bp;

Composition 321 A; 235 C; 261 G; 209 T; 0 OTHER

Percentage: 31% A; 23% C; 25% G; 20% T; 0% OTHER

Molecular Weight (kDa): ssDNA: 317.79 dsDNA: 632.5

ORIGIN

   1 ATGGTGCGCG GTAAGCCTAT CTCTAAGGAG ATCAGAGTAC TGATCAGGGA CTATTTTAAG  61 TCTGGGAAGA CACTCACTGA GATAAGCAAG CAGTTAAACT TGCCTAAGAG CTCTGTGCAT 121 GGGGTGATAC AGATTTTCAA GAAAAATGGG AACATTGAGA ATAACATCGC GAATAGAGGC 181 CGAACATCCG CAATAACCCC CCGCGACAAG AGACAGCTGG CCAAAATTGT GAAGGCTGAC 241 CGCCGCCAAT CCCTGAGAAA CTTGGCTTCC AAGTGGTCGC AGACCATTGG CAAGACTGTC 301 AAGCGGGAGT GGACCCGGCA GCAATTAAAG AGTATTGGCT ACGGTTTTTA TAAGGCCAAG 361 GAAAAACCCC TGCTTACGCT TCGGCAAAAA AAGAAGCGTC TGCAATGGGC TCGGGAAAGG 421 ATGTCTTGGA CTCAAAGGCA GTGGGATACC ATCATCTTCA GCGATGAGGC TAAATTTGAT 481 GTGAGTGTCG GCGACACGAG AAAGCGCGTC ATCCGTAAGA GGTCCGAGAC ATACCATAAG 541 GACTGCCTGA AAAGAACAAC CAAGTTTCCT GCAAGCACTA TGGTATGGGG ATGTATGTCT 601 GCCAAAGGAC TCGGAAAGCT TCACTTCATC GAAGGGACCG TTAATGCCGA AAAATACATT 661 AACATTCTCC AGGATAGTTT GCTGCCCTCA ATACCAAAAC TATCCGATTG TGGTGAATTC 721 ACTTTTCAGC AGGACGGAGC ATCATCGCAC ACCGCCAAGC GGACCAAAAA CTGGCTGCAG 781 TACAATCAGA TGGAGGTGCT CGATTGGCCC TCAAATAGTC CGGATCTAAG CCCAATCGAA 841 AATATCTGGT GGCTAATGAA AAACCAGCTG CGAAACGAGC CACAGAGGAA CATTTCCGAC 901 TTGAAAATCA AGCTGCAAGA GATGTGGGAC TCAATCTCTC AGGAGCACTG CAAAAACCTG 961 CTCAGCAGCA TGCCTAAACG AGTGAAATGC GTGATGCAGG CCAAGGGCGA CGTTACACAG1021 TTCTGA

This sequence corresponds to the normal mammalian codon usage andresults in a protein sequence after translation that is identical to thefly transposase protein sequence. The gene (cDNA) was synthesized fromoverlapping oligonucleotides in three parts (4 for part A and B and 6for part C, see below) (upper, sense strand, lower antisense strandoverlapping oligonucleotides are shown in bold). Each part was filled inby polymerase, ligated and then amplified by PCR using the outer 5′oligonucleotides of each DNA strand, and cloned. The three parts weresubsequently put together in one cDNA by standard ligation and cloning:

-   -   part A plus the Kozak sequence preceding the start codon

Linker A Kozak: CCACCATGG

     AatII    NcoI −15 CCCCGACGTCCCACCATGGTGCGCG GTAAGCCTAT CTCTAAGGAGATCAGAGTAC TGATCAGGGA CTATTTTAAG                TACCACGCGC CATTCGGATAGAGATTCCTC TAGTCTCATG ACTAGTCCCT GATAAAATTC 61                TCTGGGAAGACACTCACTGA GATAAGCAAG CAGTTAAACT TGCCTAAGAG CTCTGTGCAT               AGACCCTTCT GTGAGTGACT CTATTCGTTC GTCAATTTGA ACGGATTCTCGAGACACGTA 121                GGGGTGATAC AGATTTTCAA GAAAAATGGGAACATTGAGA ATAACATCGC GAATAGAGGC                CCCCACTATG TCTAAAAGTTCTTTTTACCC TTGTAACTCT TATTGTAGCG CTTATCTCCG 181               CGAACATCCG CAATAACCCC CCGCGACAAG AGACAGCTGG CCAAAATTGTGAAGGCTGAC                GCTTGTAGGC GTTATTGGGG GGCGCTGTTC TCTGTCGACCGGTTTTAACA CTTCCGACTG 241                CGCCGCCAAT CCCTGAGAAACTTGGCTTCC AAGTGGTCGC AGACAATTCG CAAGACTGTC                GCGGCGGTTAGGGACTCTTT GAACCGAAGG TTCACCAGCG TCTGTTAACC TGATCAGGGG                                                              MfeI    Spel

Linker B

                                                MfeI                                           GGGGCAATTGG CAAGACTGTCGCGGCGGTTA GGGACTCTTT GAACCGAAGG TTCACCAGCG TCTGTTAACC GTTCTGACAG 301AAGCGGGAGT GGACCCGGCA GCAATTAAAG AGTATTGGCT ACGGTTTTTA TAAGGCCAAGTTCGCCCTCA CCTGGGCCGT CGTTAATTTC TCATAACCGA TGCCAAAAAT ATTCCGGTTC 361GAAAAACCCC TGCTTACGCT TCGGCAAAAA AAGAAGCGTC TGCAATGGCC TCGGGAAAGGCTTTTTGGGG ACGAATGCGA AGCCGTTTTT TTCTTCGCAG ACGTTACCCG AGCCCTTTCC 421ATGTCTTGCA CTCAAACGCA GTGGGATACC ATCATCTTCA GCGATGAGGC TAAATTTGATTACAGAACCT GAGTTTCCGT CACCCTATGG TAGTAGAAGT CGCTACTCCG ATTTAAACTA 461GTGAGTGTCG GCGACACGAG AAAACGCGTC ATCCGTAAGA GGTCCGAGAC ATACCATAAGCACTCACAGC CGCTGTGCTC TTTTGCGCAG TAGGCATTCT CCAGGCTCTG TATGGTATTC 541GACTGCCTGA AAAGAACAAC CAAGTTTCCT GCAAGCACTA TGGTATGGGG ATGTATGTCTCTGACGGACT TTTCTTGTTG GTTCAAAGGA CGTTCGTGAT ACCATACCCC TACATACAGA 601GCCAAAGGAC TCGGAAAGCT TCACTTCATC GAAGGGACCG TTAATGCCGA AAAATACATTCGGTTTCCTG AGCCTTTCGA ACCCCTACGTACCCC        HindIII        NsiI

Linker C

               HindIII 601             CCCCAAGCT TCACTTCATC GAAGGGACCGTTAATGCCGA AAAATACATT                 TTCGA AGTGAAGTAG CTTCCCTGGCAATTACGGCT TTTTATGTAA 661 AACATTCTCC AGGATAGTTT GCTGCCCTCA ATACCAAAACTATCCGATTG TGGTGAATTC TTGTAAGAGG TCCTATCAAA CGACGGGAGT TATGGTTTTGATAGGCTAAC ACCACTTAAG 721 ACTTTTCAGC AGGACGGAGC ATCATCGCAC ACCGCCAAGCGGACCAAAAA CTGQCTGCAG TGAAAAGTCG TCCTGCCTCG TAGTAGCGTG TGGCGGTTCGCCTGGTTTTT GACCGACGTC 781 TACAATCAGA TGGAQGTGCT CGATTGGCCC TCAAATAGTCCGGATCTAAG CCCAATCGAA ATGTTAGTCT ACCTCCACGA GCTAACCGGG AGTTTATCAGGCCTAGATTC GGGTTAGCTT 841 AATATCTGGT GGCTAATGAA AAACCAGCTG CGAAACGAGCCACAGAGGAA CATTTCCGAC TTATAGACCA CCGATTACTT TTTGGTCGAC GCTTTGCTCGGTGTCTCCTT GTAAAGGCTG 901 TTGAAAATCA AGCTGCAAGA GATGTGGGAC TCAATCTCTCAGGAGCACTG CAAAAACCTG AACTTTTAGT TCGACGTTCT CTACACCCTG AGTTAGAGAGTCCTCGTGAC GTTTTTGGAC 961 CTCAGCAGCA TGCCTAAACG AGTGAAATGC GTGATGCAGGCCAAGGGCGA CGTTACACAG GAGTCGTCGT ACGGATTTGC TCACTTTACG CACTACGTCCGGTTCCCGCT GCAATGTGTC 1021 TTCTGAGCAT CC AAGACTCCTA GGCCCCGGGGAGATCTCCTA CGTAGGGG      BamHI             XbaI    NsiI

Materials and Methods Plasmid Construction

The construction of the modified Minos transposon pMiCMVGFP (FIG. 5),which was used for the generation of transgenic mice, is described inExample 1. In short, a 2.2 kb fragment, containing a humanised GFP genedriven by a CMV promoter and followed by an intervening sequence and anSV40 poly A signal, was positioned between Minos inverted repeats. A loxP site was included in front of the left inverted repeat for thegeneration of single copy transgenic animals if needed.

The Minos transposase cDNA was cloned as a 1 kb ClaI/NotI fragment inthe vector Pev3 (Clare Gooding, Biotechnology Dept, Zeneca,Macclesfield, UK; Pev3 is further described in Needham et al., Nucl.Acids Res., 20, 997-1003, 1992) A 3.8 kb ClaI/Asp718 fragment from theresulting plasmid (containing the Minos transposase cDNA followed by anintron and a polyadenylation signal from the human β globin gene) wascloned in pBluescript SK ⁺(Stratagene, La Jolla, Calif., USA) creatingthe plasmid pBlue/ILMi/3′β. A 6.5 kb blunt Asp718 fragment from plasmidZP3/6.5Luc (Lira, S. et al (1990) Proc. Natl. Acad. Sci. USA 87,7215-7219.) containing the 5′ flanking region and promoter of the zonapellucida 3 (ZP3) gene was cloned into the EcoRV site of pBlue/ILMi/3′β,resulting in plasmid ZP3/ILMi (FIG. 6), which was used for thegeneration of transgenic mice expressing the transposase in developingeggs.

Generation of Transgenic Mice

To generate Minos transposase expressing lines, a 10.3 kb SmaI/Asp718fragment was excised from pZP3/ILMi (FIG. 6), separated from plasmidsequences by gel electrophoresis (Sambrook, J et al Molecular Cloning. ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), purified and concentrated using an ELUTIP-d column(Schleicher & Schuell GmbH, Dassel, Germany) and injected intofertilised oocytes (FVB×FVB) at a concentration of 4 ng/μl. Injectedeggs were transferred into pseudopregnant mice and transgenic offspringwas identified by Southern blot analysis of tail DNA (Southern, E. M.(1975). J. Mol. Biol. 98, 503-517).

The transposon carrying (MCG) line was generated as described above andin (Zagoraiou, L et al (2001) Proc. Natl. Acad. Sci. USA 98,11474-11478).

RT-PCR

For RT-PCR analysis, total RNA was isolated from different organs ofZP3/ILMi transgenic mice using the Ultraspec RNA isolation system(Biotech Laboratories, Houston, Tex., USA). From 1 μg of total RNA, cDNAwas synthesised in a 20 μl reaction using Reverse Transcriptase (SuperRT; HT Biotechnology, Cambridge, UK) and oligo(dT) primer. PCR reactionswere performed in a volume of 50 μl PCR buffer (Life Technologies,Paisley, UK) containing 1 μl of the cDNA from the RT reaction, 1.5 mMMgCl₂, 100 ng of each primer, 0.2 mM dNTPs and 2.5 U Taq DNA polymerase(Pharmacia). A total number of 25 cycles were performed withdenaturation at 94° C. for 45 seconds, annealing at 55° C. for 30seconds and extension at 72° C. for 45 seconds. PCR products werevisualised by electrophoresis on a 2% agarose gel. The Minos transposasespecific primers Minos1: 5′-CAGCTTCGAAATGAGCCAC-3′ and beta EX:5′-TGGACAGCAAGAAAGCGAG-3′ were used. Primers specific for murinehypoxanthine phosphoribosyltransferase (HPRT) were:5′CACAGGACTAGAACACCTGC-3′ and 5′-GCTGGTGAAAAGGACCTCT-3′.

Breeding Program

Transposon carrying (MCG) females were bred with ZP3/ILMi line 15 males.Double positive females obtained from these crosses were bred with wildtype (WT) males and their offspring analysed by Southern blot analysisfor possible transposition events. Genomic DNA was digested either withEcoRV or BglII, separated on a 0.7 or 1% agarose gel (Sigma, Steinheim,Germany), blotted onto a nylon membrane (Hybond-N⁺, Amersham Pharmacia,Buckinghamshire England) and probed with ³²P labelled 737 bp SacI/NotIGFP fragment from pMiCMVGFP.

DNA Fluorescent in Situ Hybridisation (FISH) Analysis

Mouse metaphase spreads were prepared according to routine proceduresfrom peripheral white blood cells (Mulder, M. P et al. (1995). HumGenet. 96(2):133-141). The 737-bp SacI/NotI GFP fragment from thepMiCMVGFP construct was used as a probe. The probe was either labelledwith biotin (Boehringer Mannheim) and immunochemically detected directlywith FITC or a tyramide based step was included to improve signaldetection (Raap, A. K. et al (1995) Human Molecular Genetics 4,529-534). The DNA was counterstained with DAPI.

Cloning of the Insertion Sites

Mouse DNA from animals with a new Minos insertion site was cut withEcoRV or BglII and resolved in a 0.7% agarose gel. The gel regionscontaining transposition events were cut out and the DNA was isolated.Depending on the fragment size, inverse PCR was performed eitherdirectly on self-ligated fragments using Minos primers Imio1 (5′AAGAGAATAAAATTCTCTTTGAGACG 3′) for the first PCR and IMio2 (5′GATAATATAGTGTGTTAAACATTGCGC 3′) for the nested PCR (Klinakis, A. G.,Zagoraiou, L., Vassilatis, D. K & Savakis, C. (2000) EMBO Reports 11,416-421.), or the obtained EcoRV or BglII fragments were furtherdigested with AluI and then circularised. Inverse PCR was performed withMinos primers Imio1 and Imii1 (5′ CAAAAATATGAGTAATTTATTCAAACGG 3′),followed by nested PCR 20 with primers IMio2 and IMii2 (5′GCTTAAGAGATAAGAAAAAAGTGACC 3′) as previously described (Klinakis, A. G.,Zagoraiou, L., Vassilatis, D. K & Savakis, C. (2000) EMBO Reports 11,416-421). In this way, left and right flanks were amplified separately.The PCR fragments were either sequenced directly or after cloning intothe pGEM T easy vector (Promega), or PCRII vector (Invitrogen). With thesequences obtained, a BLAST search was performed against the mousegenome sequences available at the time in the Celera (www.celera.com)database.

Results

The transposon carrying transgenic mouse line (MCG) was generated. Itcontains 6 copies of Minos transposon MiCMVGFP (FIG. 5) integrated inmouse chromosome 14. The transposon is nonautonomous, i.e. it cannottranspose on its own, since it lacks the transposase gene. In parallel,two transposase expressing mouse lines were generated. These expressedthe Minos transposase specifically in growing oocytes due to the use ofa 6.5 Kb 5′ flanking region and promoter of the ZP 3 gene (FIG. 6). Inboth of the Minos transposase (ZP3/ILMi) lines, the transgene integratedas a tandem array. In this example we used ZP3/I LMi line 15 with thehigher number of copies integrated (data not shown). As expected,transposase expression in this line was restricted to the ovaries (FIG.7). RT-PCR performed on RNA samples from different tissues of transgenicZP3/ILMi line showed that the ˜360 bp fragment, corresponding to thecorrectly spliced transposase RNA, was restricted to the ovary. Theamplification of contaminating DNA to a similar sized fragment wasprevented by using a primer which spans an exon/intron junction (betaglobin IVSII—FIG. 6).

Since the Minos transposase expression from the transgene is driven bythe ZP3 promoter, it should be expressed only in growing oocytes duringa 2-to-3-week period of oogenesis.

Normally, zona pellucida transcripts cannot be detected in primordialoocytes (10-15 μm), and maximum levels are observed in 50 μm-diameteroocytes. As the oocytes reach maximum size (70-80 μm), the level of ZP3transcripts begins to decline. Ovulated eggs contain less than 5% of thepeak levels of all zona pellucida transcripts (Millar, et al. (1991)Molecular and Cellular Biology 11, 6197-6204; Liu, C et al. (1996) Proc.Natl. Acad. Sci. USA., 5431-5436). To exclude the possibility that sometransposase activity remains in mature oocytes to mediate transpositionof a paternally contributed transposon transgene, the Zp3/ILMi males(that do not express the transposase) were mated to females of themultycopy transposon carrying line MCG. Female progeny positive for bothtransgenes were selected for further study. We analysed 307 progeny ofthe double positive females and wild type males by Southern blotanalysis. EcoRV and/or BglII digested tail DNA was blotted andhybridised with the GFP probe. Since neither of these two enzymes cutswithin the transposon, there is one single band that hybridises to thetransposon probe in an MCG line. If transposition occurs and thetransposon inserts outside the genomic EcoRV or BglII fragment where itwas initially present, a new band will be detected after hybridisationwith the transposon probe. Out of 307 mice, 146 mice were transposon(GFP) positive. Among these 146 mice, 12 transposition events wereobserved that resulted in a novel restriction fragment on the blotgiving a transposition frequency of 8.2%. In two mice, two independenttransposition events were found (FIG. 8 lanes 2 and 5). The observationthat some offspring carry Minos mediated insertions but did not inheritthe transposase transgene strongly suggests that transposition occurredin the germ cells of the mother prior to meiosis II.

To prove that the observed transposition events had indeed occurred inthe germ line, animals with transposition events were further crossed towild type mice. Analysis of their progeny showed that these mice stablytransmitted the reinserted Minos element (FIG. 8). The segregation of atransposon concatemer and a new insertion into two different lines inthe F1 generation was clear evidence that transposition had occurred toanother chromosome (FIG. 8, lane 2 versus 3 and 4, and lane 8 versus 9).In all transposition events except one, a single copy of the transposonwas mobilised.

It has been noted previously that the size of a transposon influencesits transposition frequency; longer elements tend to transpose with alower frequency (Lampe, D. J. et al (1998). Genetics 149, 179-187;Fischer, S. E., et al. (1999) Mol. Gen. Genet. 262, 268-274.),suggesting that when transposase binding sites (inverted repeats) arecloser to each other they are recognised more efficiently.

Minos transpositions are characterised by a precise integration of theelement without mobilisation of flanking DNA. In Drosophila and in HeLacells, the transposon inserts into TA dinucleotide causing target siteduplication upon insertion (17, 29). To investigate the structure of theinsertions in the mouse genome, we cloned the flanking regions (asdescribed in Materials and Methods) from five different transpositionevents. As is observed in Drosophila, the Minos ends were flanked by thediagnostic TA dinucleotide followed by sequences unrelated to thesequence that flanks the element in the founder mouse line MCG (FIG. 9).BLAST searches with the obtained sequences in the Celera mouse genomedatabase showed that all five of the novel flanking sequences (in onecase only one flanking sequence was obtained) correspond to widelyscattered genomic locations (FIG. 9). Out of five transposition eventsanalysed only one was on chromosome 14. It is a single copy of thetransposon integrated into a centromeric region without the presence ofa transposon concatamer on tip of chromosome 14. Thus this transpositionevent occurred into a different chromosome (See FIG. 10). The DNAFluorescent in Situ Hybridisation (FISH analysis) performed on metaphasespreads from peripheral white blood cells confirmed the results obtainedfrom sequencing of the flanking regions (data not shown). However,transposition events that were clearly detectable by Southern blotanalysis but were located close to the original site on the samechromosome would not be identified by FISH. It is actually verydifficult to detect single copy (3 kb) transpositions by FISH and thismay partially explain the low frequency of transposition (0.61%) weobtained previously with the same transposon containing transgenic line(MCG) and Minos transposase expressed specifically in T cells(Zagoraiou, L., et al. (2001) Proc. Natl. Acad. Sci. USA 98,11474-11478) where FISH was the only method of detection used. Since“local insertions”, close to the original site of the element, are quitefrequent at least with the Drosophila P element (Zhang, P. & Sprading,A. C. (1993) Genetics 133, 361-373) and Sleeping Beauty (Luo, G. et al.(1998) Proc. Natl. Acad. Sci. USA. 95, 10769-10773; Fisher, S. E. J., etal. (2001) Proc. Natl. Acad. Sci. USA. 98, 6759-6764.), it is unlikelythat this would not be the case with the Minos element.

In order to determine the size of the EcoRV/BglII flanking regions wegenerated a single copy transposon line from the MCG line using theloxP/Cre system (data not shown). On the basis of Southern blot data,there is approximately 10 kb of genomic sequence flanking thetransposon, within the EcoRV and BglII diagnostic digests. Localreinsertions that occur within this area (but which are not interestingfor mutagenesis purposes), will escape detection and are not included inour estimation of the frequency of transposition. Thus, the frequency of8.2% represents the “useful” transposition frequency rather than theactual transposition frequency. The reported frequency of transpositionsin the mouse male germ line with Sleeping Beauty was approximately 20%,but only 2% had transposed to a different chromosome (Fisher, S. E. J.,Wienholds, E. & Plasterk, R. H. A. (2001) Proc. Natl. Acad. Sci. USA.98, 6759-6764). In contrast we find for Minos approx. 6% transpositionto a different chromosome (8 out of 11 analysed and 1 unknown, out of146 offspring), while only 3 out of 11 transpositions were to the samechromosome (e.g. FIG. 10B). This suggests that the Minos system has apreference for transpositions to a different chromosome (provided thereweren't many transpositions close to the original site that wentundetected). It should be noted however that a direct comparison of thedifferent transposon systems published to date may not be very valid.The size, the copy number and the initial chromosomal position of thetransposon might all affect the transposition efficiency and none ofthese were comparable in the different systems.

In conclusion, these results show that transposition can be achieved inthe mouse germ line and that, by selecting the time of induction,mobilisation of transposons may be induced at a predetermined stage ofembryo development. Furthermore, the results demonstrate that systemsusing, for example, Minos, are excellent tools for insertional geneinactivation, gene tagging, enhancer trapping and exon trapping inorganisms, for example, mice.

Example 6 Generation of Transgenic Progeny and Induction ofTransposition Events

Transposition events were detected in the progeny of a cross between adouble positive transposon/transposase transgenic male and anon-transgenic female.

Double positive transposon/transposase transgenic males were obtained bycrossing Trap A or Trap B transgenic lines with an H1 t transposaseknock in line (see FIG. 16). Trap A and Trap B transgenic lines weregenerated by means of transgenesis (injection of the DNA into fertilizedmouse oocytes). The injected construct is depicted in FIG. 16. The TrapA line contains approximately 30 copies of the transposon (trapconstruct) integrated on chromosome 5 and the Trap B line hasapproximately 40 copies of the transposon integrated on chromosome 4. AnH1 t knock in line was generated by blastocyst injection of ES cellswhereby mousified Minos transposase (see FIG. 17) was targeted into theH1-t locus by homologous recombination, thereby ensuring sperm specificexpression. Southern blotting demonstrated the correct ES clone (seeFIG. 16). The resulting chimeras gave germ line transmission. The linewas further bred to a Rosa FLP mouse to remove the puro gene. Thetargeting strategy and the removal of the Puro gene are presented inFIG. 16.

Transpostion positive offspring were produced by crossing the doublepositive transposon/transposase transgenic males (described above) withnon-transgenic females, as depicted in FIG. 18. Transposition eventsoccurred in the sperm.

FIGS. 19 and 20 are Southern blot analyses showing transposition eventsin litters 04-11642 and 04-11761 (produced by breeding a non-transgenicfemale with male 03-23712-05 (transgenic for Trap A and transposase));litters 04-11624 and 04-11737 (produced by breeeding a non-transgenicfemale with male 03-23830-07 (transgenic for Trap B and transposase));and litters 04-12496 and 04-15687 (produced by breeding a non-transgenicfemale with male 03-23830-07 (transgenic for Trap B and transposase).The results of FISH analysis of Minos transposition events are presentedin FIGS. 21 and 22.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are apparent to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1. A method of generating a non-human mammalian transgenic embryo oroffspring by germ-line transmission wherein one or more insertionalevents are present in all cells and tissues of said embryos oroff-spring comprising: a) Generating either (i) a first non-humantransgenic adult male mammal wherein transposon transposition occurs indeveloping spermatocytes by the regulated expression of a cognatetransposase such that the resultant sperm carry one or more insertionalevents, or (ii) a first non-human transgenic female mammal whereintransposon transposition occurs in developing oocytes such that theresultant oocytes carry one or more insertional events: b) Crossingeither (i) the first non-human transgenic adult male with a secondnon-human adult female mammal lacking a transposon and cognatetransposase activity, or (ii) the first non-human transgenic adultfemale with a second non-human adult male lacking a transposon andcognate transposase activity to provide: Non-human mammalian embryos oroffspring comprising one or more insertional events in all cells andtissues contributed either by sperm or oocyte derived from either (i)the non-human transgenic adult male mammal, or (ii) the non-humantransgenic adult female mammal of step (a).
 2. The method of claim 1wherein transposase activity in developing sperm is regulated by H1tregulatory sequences.
 3. The method of claim 1 wherein transposaseactivity in the developing oocyte is regulated by ZP3 regulatorysequences
 4. The method of claim 1, 2, or 3, wherein one or both of thefirst or second non-human adult transgenic mammals is prone to a diseasestate
 5. The method of claim 1 wherein the second non-human adult mammalis generated by germ-line transmission
 6. The method of claim 1 whereinas a result of a transposition event there is a measurable change in aphenotypic characteristic in any non-human embryo or progeny relative tothe first or second adult non-human mammals
 7. The method of claim 6wherein said change in phenotype is correlated with a transpositionevent by a method comprising: (a) identifying in the transgenic embryoor offspring a variant phenotype; (b) detecting the position of one ormore transposon transposition events in the genome of one or more ofsaid cells; and (c) correlating the position of the transposition eventswith the observed variant phenotype, the position of the transpositionevents being indicative of the location of one or more genetic lociassociated with the observed variant phenotype.
 8. A method according toclaim 6 wherein a transposition associated with a disease relatedphenotype is identified by a method comprising: (b) identifying in thetransgenic embryo or offspring a disease related variant phenotype; (c)detecting the position of one or more transposon transposition events inthe genome of one or more of said cells; and (d) correlating theposition of the transposition events with the observed variantphenotype, the position of the transposition events being indicative ofthe location of one or more genetic loci associated with the observedvariant phenotype.
 9. The method of claim 7 or 8, further comprising thestep of sequencing genomic DNA adjacent to said transpositions, for theidentification of one or more disease associated genes or regulatorysequences.
 10. A method for producing a library of transgenic embryos oroffspring comprising generating a plurality of transgenic embryos andinducing transposition therein by the method according to claim
 6. 11. Alibrary of transgenic non-human transgenic mammals produced according tothe method of claim
 10. 12. A method according to claim 9 where the stepof sequencing genomic DNA adjacent to said transpositions occurs priorto phenotypic analysis of embryos or progeny
 13. A library of transgenicnon-human transgenic mammals generated according to claim 12 comprisingoff-spring each comprising a gene disruption in a different gene. 14.The method according to claim 1, wherein the transposon comprises anucleic acid sequence encoding a selectable marker.
 15. The methodaccording to claim 14, wherein the selectable marker is a fluorescent orluminescent polypeptide.
 16. A method of claim 14 or 15 wherein theselectable marker is incorporated in an exon trap
 17. A Method of claim14 or 15 wherein the selectable marker is incorporated in an enhancertrap
 18. A method of claim 6 wherein the change in phenotype is definedby the enhanced expression of a selectable marker resulting from one ormore insertional events